Engineered amylase variants

ABSTRACT

The present invention provides engineered amylase polypeptides and compositions thereof. The engineered amylase polypeptides have been optimized to provide improved thermostability, protease stability, and stability under a range of pH conditions, including acidic (pH&lt;7) conditions. The invention also relates to the use of the compositions comprising the engineered amylase polypeptides for therapeutic and/or nutritional purposes. The present invention also provides polynucleotides encoding the engineered amylase polypeptides, as well as methods for making the engineered polynucleotides and amylase polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.provisional application No. 63/071,641, filed Aug. 28, 2020, the entirecontents of which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention provides engineered amylase polypeptides andcompositions thereof. The engineered amylase polypeptides have beenoptimized to provide improved thermostability, protease stability, andstability under a range of pH conditions, including acidic (pH<7)conditions. The invention also relates to the use of the compositionscomprising the engineered amylase polypeptides for therapeutic and/ornutritional purposes. The present invention also providespolynucleotides encoding the engineered amylase polypeptides, as well asmethods for making the engineered polynucleotides and amylasepolypeptides.

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The official copy of the Sequence Listing is submitted concurrently withthe specification as an ASCII formatted text file via EFS-Web, with afile name of “CX7-211US2_ST25.txt”, a creation date of Aug. 27, 2021,and a size of 5.61 megabytes. The Sequence Listing filed via EFS-Web ispart of the specification and is incorporated in its entirety byreference herein.

BACKGROUND OF THE INVENTION

Pancreatic enzyme replacement therapy (PERT) finds use in the treatmentof pancreatic enzyme insufficiency (PEI). Various disorders, includingpancreatitis, cystic fibrosis, celiac disease, inflammatory boweldisease, and pancreatic cancer can lead to PEI, as a consequence ofdecreased secretion of pancreatic enzymes into the duodenum. Thisresults in poor digestion of food, inadequate absorption of fat,proteins, carbohydrates, and vitamins by the intestines, which can leadto malnutrition. Although orally administered PERT treatments arecurrently available, the condition may not be alleviated in some people,due to insufficient activity of the PERT in the gastrointestinal tractand/or insufficient patient compliance with the therapy due to thesignificant pill burden associated with current treatment protocols. Insome cases, the coefficient of fat absorption (CFA) and/or coefficientof nitrogen absorption (CNA) is inferior to that of healthy patients,resulting in weight loss and other health concerns. Thus, a need remainsin the art for improved PERT treatments.

SUMMARY OF THE INVENTION

The present invention provides engineered amylase polypeptides andcompositions thereof. The engineered amylase polypeptides have beenoptimized to provide improved thermostability, protease stability, andstability under a range of pH conditions, including acidic (pH<7)conditions. The invention also relates to the use of the compositionscomprising the engineered amylase polypeptides for therapeutic and/ornutritional purposes. The present invention also providespolynucleotides encoding the engineered amylase polypeptides, as well asmethods for making the engineered polynucleotides and amylasepolypeptides.

The present invention provide recombinant amylases and/or biologicallyactive recombinant amylase fragments comprising amino acid sequencescomprising at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 91%, at leastabout 92%, at least about 93%, at least about 94%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, or at leastabout 99% sequence identity to SEQ ID NO: 2. In some embodiments, therecombinant amylase and/or biologically active recombinant amylasefragment comprises a polypeptide sequence having at least about 70%,about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%,about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about94%, about 95%, about 96%, about 97%, about 98%, about 99%, or moresequence identity to SEQ ID NO: 2, 18, 40, 156, 174, 276, 308, 422, 456,546, 632, 664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494, 1580,1612, and/or 1728.

The present invention provide recombinant amylases and/or biologicallyactive recombinant amylase fragments comprising amino acid sequencescomprising at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 2. In some embodiments, the recombinantamylase and/or biologically active recombinant amylase fragmentcomprises a polypeptide sequence having at least 70%, 75%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ormore sequence identity to SEQ ID NO: 18, 40, 156, 174, 276, 308, 422,456, 546, 632, 664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494,1580, 1612, and/or 1728.

In some embodiments, the recombinant amylase comprises a polypeptidesequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO: 2 or 18, or a functional fragment thereof, and wherein therecombinant amylase comprises one or more substitutions at positionsselected from 2, 6, 16, 18, 19, 24, 26, 27, 29, 31, 33, 37, 38, 39, 41,45, 48, 49, 51, 52, 58, 61, 66, 72, 73, 74, 81, 82, 91, 92, 94, 95, 98,103, 111, 112, 114, 122, 123, 124, 126, 127, 128, 129, 130, 131, 132,134, 137, 140, 141, 145, 149, 157, 158, 165, 166, 167, 170, 171, 172,178, 180, 183, 184, 189, 194, 203, 204, 205, 206, 210, 214, 216, 220,221, 223, 224, 225, 226, 227, 228, 246, 250, 252, 253, 254, 255, 256,257, 258, 259, 268, 269, 270, 272, 275, 276, 279, 280, 282, 284, 285,286, 294, 295, 298, 301, 302, 304, 305, 306, 308, 309, 310, 313, 315,317, 319, 322, 324, 331, 338S, 345, 346, 349, 359, 360, 366, 371, 374,380, 383, 384, 385, 387, 388, 392, 393, 394, 396, 397, 409, 411, 412,413, 415, 417, 420, 423, 428, 429, 431, 437L 441, 442, 444, 446, 448,451, 455, 458, 459, 463, 464, 466, 467, 468, 469, 470, 473, 474, 480,481, 482, 483, 491, 492, 493, 494, and 495, wherein the amino acidpositions of the polypeptide sequence are numbered with reference to SEQID NO: 2 or 18.

In some additional embodiments, the recombinant amylase comprises apolypeptide sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequenceidentity to SEQ ID NO: 2 or 18, or a functional fragment thereof, andwherein the recombinant amylase comprises one or more substitutions atpositions selected from 16, 26, 29, 33, 91, 114, 157, 158, 180, 184,214, 223, 256, 272, 298, 302, 371, 380, 383, 394 412, 428, and 437,wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 2 or 18.

In some additional embodiments, the recombinant amylase comprises apolypeptide sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequenceidentity to SEQ ID NO: 2 or 18, or a functional fragment thereof, andwherein the recombinant amylase comprises one or more substitutionsselected from 16D, 26A, 29G, 29R, 29S, 29A, 29E, 33E, 91H, 114H, 157Y,158R, 180E, 180Q, 184D, 184K, 214G, 223L, 256T, 272G, 272K, 272P, 272R,272S, 298H, 302Q, 302G, 302H, 302R, 302V, 302W, 371F, 380R, 383E, 383I,394N, 394S, 394G, 412A, 412L, 412D, 412S, 428T, and 437L, wherein theamino acid positions of the polypeptide sequence are numbered withreference to SEQ ID NO: 2 or 18.

In some additional embodiments, the recombinant amylase comprises apolypeptide sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequenceidentity to SEQ ID NO: 2 or 18, or a functional fragment thereof, andwherein the recombinant amylase comprises at least one substitution orsubstitution set at one or more positions selected from 2/73/256/493,16/33/73/130/256/282/371/374/493, 16/33/130/256/302/371/383/470,16/33/157/256/302/371/383, 16/73/74/256/282/366/371/383/493,16/73/256/282/302/331/371/383, 16/130/256/276/374/470, 16/256/302/374,19/72/345/473, 26/27/396/412/428/473, 26/27/412, 26/27/466/473, 29,73/130/256/282/302/493, 74/371/374/383/493, 130/256/493, 149, 158, 254,256, 256/383, 257, 259, 272, 279, 284, 317/383, 322, 345/388/396/428,388/396/412/428, 396/412, 396/412/428/466, 409, 412, and 495, whereinthe amino acid positions of the polypeptide sequence are numbered withreference to SEQ ID NO: 2 or 18. In some further embodiments, therecombinant amylase comprises a polypeptide sequence having at least70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 2 or 18, ora functional fragment thereof, and wherein the recombinant amylasecomprises at least one substitution or substitution set selected from2K/73V/256S/493D, 16D/33E/73V/130D/256S/282D/371F/374S/493D,16D/33E/130D/256S/302Q/371F/383E/470T, 16D/33E/157Y/256S/302Q/371F/383E,16D/73V/74A/256S/282D/366L/371F/383E/493D,16D/73V/256S/282D/302Q/331P/371F/383E, 16D/130D/256S/276Q/374S/470T,16D/256S/302Q/374S, 19E/72T/345A/473D, 26A/27L/396H/412D/428E/473D,26A/27L/412D, 26A/27L/466E/473D, 29G, 29R, 29S,73V/130D/256S/282D/302Q/493D, 74A/371F/374S/383E/493D, 130D/256S/493D,149R, 149T, 158R, 254G, 254S, 256S, 256S/383E, 257E, 257G, 257R, 257S,257V, 259Q, 272G, 272K, 272P, 272R, 272S, 279R, 284C, 284R, 284S, 284Y,317G/383L, 322A, 345A/388E/396H/428E, 388E/396H/412D/428E, 396H/412D,396H/412D/428E/466E, 409S, 412S, 495E, 495L, and 495Y, wherein the aminoacid positions of the polypeptide sequence are numbered with referenceto SEQ ID NO: 2 or 18. In some further embodiments, the recombinantamylase comprises a polypeptide sequence having at least 70%, 75%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or more sequence identity to SEQ ID NO: 2 or 18, or a functionalfragment thereof, and wherein the recombinant amylase comprises at leastone substitution or substitution set selected from R2K/I73V/A256S/E493D,E16D/D33E/I73V/E130D/A256S/E282D/Y371F/H374S/E493D,E16D/D33E/E130D/A256S/M302Q/Y371F/Q383E/V470T,E16D/D33E/F157Y/A256S/M302Q/Y371F/Q383E,E16D/I73V/P74A/A256S/E282D/F366L/Y371F/Q383E/E493D,E16D/I73V/A256S/E282D/M302Q/H331P/Y371F/Q383E,E16D/E130D/A256S/T276Q/H374S/V470T, E16D/A256S/M302Q/H374S,K19E/S72T/P345A/E473D, E26A/R27L/D396H/L412D/D428E/E473D,E26A/R27L/L412D, E26A/R27L/Q466E/E473D, D29G, D29R, D29S,I73V/E130D/A256S/E282D/M302Q/E493D, P74A/Y371F/H374S/Q383E/E493D,E130D/A256S/E493D, Q149R, Q149T, K158R, E254G, E254S, A256S,A256S/Q383E, P257E, P257G, P257R, P257S, P257V, P259Q, D272G, D272K,D272P, ddD272R, D272S, D279R, K284C, K284R, K284S, K284Y, V317G/Q383L,F322A, P345A/D388E/D396H/D428E, D388E/D396H/L412D/D428E, D396H/L412D,D396H/L412D/D428E/Q466E, V409S, L412S, 1495E, I495L, and I495Y, whereinthe amino acid positions of the polypeptide sequence are numbered withreference to SEQ ID NO: 2 or 18.

In some additional embodiments, the recombinant amylase comprises apolypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO: 40 or 988, or a functional fragment thereof, and whereinthe recombinant amylase comprises at least one substitution orsubstitution set at one or more positions selected from26/29/158/272/412, 26/29/272/279/412/493, 26/29/272/396/412,26/29/279/412/428, 26/158/272/279/388/412/428, 26/158/272/412,26/272/279/412/428/493, 29/272/279/396/412, 130/149/254/284/322/409/495,130/149/257/284/322/409/466/473, 130/254/284/322/374/409/466,130/257/284/374/409/466, 130/284/322/374/409/473,149/257/284/322/409/466, 158/272/279/394/396/412/428/493,158/272/388/412/428/493, 158/272/412/428, 158/279/388/396/412/428,254/257/284/322/409/473, 254/284/409/466/495, 272/279/412/428/493,272/412/428, 272/412/493, and 284/322/409/473, wherein the amino acidpositions of the polypeptide sequence are numbered with reference to SEQID NO: 40 or 988. In some further embodiments, the recombinant amylasecomprises a polypeptide sequence having at least 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NO: 40 or 988, or a functional fragmentthereof, and wherein the recombinant amylase comprises at least onesubstitution or substitution set selected from 26A/29R/158R/272G/412D,26A/29R/272G/279R/412D/493D, 26A/29R/272G/396H/412D,26A/29R/279R/412D/428E, 26A/158R/272G/279R/388E/412D/428E,26A/158R/272G/412D, 26A/272G/279R/412D/428E/493D,29R/272G/279R/396H/412D, 130D/149R/254S/284S/322A/409S/495E,130D/149R/257G/284S/322A/409S/466E/473D,130D/254S/284S/322A/374S/409S/466E, 130D/257G/284S/374S/409S/466E,130D/284S/322A/374S/409S/473D, 149R/257G/284S/322A/409S/466E,158R/272G/279R/394R/396H/412D/428E/493D, 158R/272G/388E/412D/428E/493D,158R/272G/412D/428E, 158R/279R/388E/396H/412D/428E,254S/257G/284S/322A/409S/473D, 254S/284S/409S/466E/495E,272G/279R/412D/428E/493D, 272G/412D/428E, 272G/412D/493D, and284S/322A/409S/473D, wherein the amino acid positions of the polypeptidesequence are numbered with reference to SEQ ID NO: 40 or 988. In somefurther embodiments, the recombinant amylase comprises a polypeptidesequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ IDNO: 40 or 988, or a functional fragment thereof, and wherein therecombinant amylase comprises at least one substitution or substitutionset selected from E26A/D29R/K158R/D272G/L412D,E26A/D29R/D272G/D279R/L412D/E493D, E26A/D29R/D272G/D396H/L412D,E26A/D29R/D279R/L412D/D428E, E26A/K158R/D272G/D279R/D388E/L412D/D428E,E26A/K158R/D272G/L412D, E26A/D272G/D279R/L412D/D428E/E493D,D29R/D272G/D279R/D396H/L412D, E130D/Q149R/E254S/K284S/F322A/V409S/1495E,E130D/Q149R/P257G/K284S/F322A/V409S/Q466E/E473D,E130D/E254S/K284S/F322A/H374S/V409S/Q466E,E130D/P257G/K284S/H374S/V409S/Q466E,E130D/K284S/F322A/H374S/V409S/E473D,Q149R/P257G/K284S/F322A/V409S/Q466E,K158R/D272G/D279R/Q394R/D396H/L412D/D428E/E493D,K158R/D272G/D388E/L412D/D428E/E493D, K158R/D272G/L412D/D428E,K158R/D279R/D388E/D396H/L412D/D428E,E254S/P257G/K284S/F322A/V409S/E473D, E254S/K284S/V409S/Q466E/1495E,D272G/D279R/L412D/D428E/E493D, D272G/L412D/D428E, D272G/L412D/E493D, andK284S/F322A/V409S/E473D, wherein the amino acid positions of thepolypeptide sequence are numbered with reference to SEQ ID NO: 40 or988.

In some additional embodiments, the recombinant amylase comprises apolypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO: 156 or 1104, or a functional fragment thereof, and whereinthe recombinant amylase comprises at least one substitution orsubstitution set at one or more positions selected from 37, 51, 91, 111,112, 114, 122, 124, 128, 140, 167, 171, 183, 194, 276, 280, 298, 397,and 464, wherein the amino acid positions of the polypeptide sequenceare numbered with reference to SEQ ID NO: 156 or 1104. In some furtherembodiments, the recombinant amylase comprises a polypeptide sequencehaving at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 156 or1104, or a functional fragment thereof, and wherein the recombinantamylase comprises at least one substitution or substitution set selectedfrom 37R, 51R, 91H, 1I1A, 112Q, 114H, 122P, 122R, 124R, 128W, 140K,167W, 171I, 183E, 183N, 183V, 194E, 194R, 276A, 276K, 280L, 298K, 298M,397T, and 464L, wherein the amino acid positions of the polypeptidesequence are numbered with reference to SEQ ID NO: 156 or 1104. In somefurther embodiments, the recombinant amylase comprises a polypeptidesequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ IDNO: 156 or 1104, or a functional fragment thereof, and wherein therecombinant amylase comprises at least one substitution or substitutionset selected from N37R, G51R, K91H, E111A, K112Q, A114H, A122P, A122R,D124R, 1128W, R140K, E167W, E171I, G183E, G183N, G183V, L194E, L194R,T276A, T276K, Q280L, E298K, E298M, E397T, and N464L, wherein the aminoacid positions of the polypeptide sequence are numbered with referenceto SEQ ID NO: 156 or 1104.

In some additional embodiments, the recombinant amylase comprises apolypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO: 174 or 1122, or a functional fragment thereof, and whereinthe recombinant amylase comprises at least one substitution orsubstitution set at one or more positions selected from 24, 91, 91/122,91/122/128/130/167/171/409, 91/122/128/130/183/322, 91/122/128/130/194,91/122/128/130/322, 91/122/128/171/183/194/322, 91/122/128/183/409,91/122/130/167/183/194/409, 91/122/130/171/183, 91/122/130/183/194,91/122/130/194, 91/122/130/194/322, 91/122/167/171/194,91/122/167/171/194/322, 91/122/171/183/194/409, 91/130/171/183/322/409,91/167/171, 91/167/183/194, 91/167/322, 91/183, 91/183/322/409, 91/194,91/322, 91/409, 122/128, 122/128/194/409, 122/130/171/322/409, 122/409,137/145/227/394/480/481, 137/145/295, 145/227/295/481, 145/309/481, 183,227, 295/394, 322, 322/409, 394, and 480/481, wherein the amino acidpositions of the polypeptide sequence are numbered with reference to SEQID NO: 174 or 1122. In some further embodiments, the recombinant amylasecomprises a polypeptide sequence having at least 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NO: 174 or 1122, or a functional fragmentthereof, and wherein the recombinant amylase comprises at least onesubstitution or substitution set selected from 24V, 91H, 91H/122P,91H/122P/130D/183N/194R, 91H/122P/130D/194R/322A, 91H/122R,91H/122R/128W/130D/167W/171I/409S, 91H/122R/128W/130D/183N/322A,91H/122R/128W/130D/194R, 91H/122R/128W/130D/322A,91H/122R/128W/171I/183N/194R/322A, 91H/122R/128W/183N/409S,91H/122R/130D/167W/183N/194R/409S, 91H/122R/130D/171I/183N,91H/122R/130D/194R, 91H/122R/167W/171I/194R,91H/122R/167W/171I/194R/322A, 91H/122R/171I/183N/194R/409S,91H/130D/171I/183N/322A/409S, 91H/167W/171I, 91H/167W/183N/194R,91H/167W/322A, 91H/183N, 91H/183N/322A/409S, 91H/194R, 91H/322A,91H/409S, 122P/409S, 122R/128W, 122R/128W/194R/409S,122R/130D/171I/322A/409S, 137A/145G/227L/394E/480P/481V, 137A/145G/295N,145G/227L/295N/481V, 145G/309S/481V, 183N, 227L, 295N/394E, 322A,322A/409S, 394E, and 480T/481V, wherein the amino acid positions of thepolypeptide sequence are numbered with reference to SEQ ID NO: 174 or1122. In some further embodiments, the recombinant amylase comprises apolypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO: 174 or 1122, or a functional fragment thereof, and whereinthe recombinant amylase comprises at least one substitution orsubstitution set selected from L24V, K91H, K91H/A122P,K91H/A122P/E130D/G183N/L194R, K91H/A122P/E130D/L194R/F322A, K91H/A122R,K91H/A122R/I128W/E130D/E167W/E171I/V409S,K91H/A122R/I128W/E130D/G183N/F322A, K91H/A122R/I128W/E130D/L194R,K91H/A122R/I128W/E130D/F322A, K91H/A122R/I128W/E171I/G183N/L194R/F322A,K91H/A122R/I128W/G183N/V409S, K91H/A122R/E130D/E167W/G183N/L194R/V409S,K91H/A122R/E130D/E171I/G183N, K91H/A122R/E130D/L194R,K91H/A122R/E167W/E171I/L194R, K91H/A122R/E167W/E171I/L194R/F322A,K91H/A122R/E171I/G183N/L194R/V409S, K91H/E130D/E171I/G183N/F322A/V409S,K91H/E167W/E171I, K91H/E167W/G183N/L194R, K91H/E167W/F322A, K91H/G183N,K91H/G183N/F322A/V409S, K91H/L194R, K91H/F322A, K91H/V409S, A122P/V409S,A122R/I128W, A122R/I128W/L194R/V409S, A122R/E130D/E171I/F322A/V409S,G137A/A145G/T227L/Q394E/F480P/C481V, G137A/A145G/K295N,A145G/T227L/K295N/C481V, A145G/T309S/C481V, G183N, T227L, K295N/Q394E,F322A, F322A/V409S, Q394E, and F480T/C481V, wherein the amino acidpositions of the polypeptide sequence are numbered with reference to SEQID NO: 174 or 1122.

In some additional embodiments, the recombinant amylase comprises apolypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO: 276 or 1224, or a functional fragment thereof, and whereinthe recombinant amylase comprises at least one substitution orsubstitution set at one or more positions selected from16/26/29/128/145/256/309/383, 16/26/33/114/145/309/383/394,16/26/33/128/130/145/302/309/322/371, 16/26/33/145/158/383/394,16/26/145/302, 16/29/33/145/256/302, 18,26/29/33/130/145/157/158/183/394, 26/29/33/145/157/183/256/309/383,26/29/122/130/145/183/256/272/309/394, 26/29/145/157/256/302/322/481,26/29/145/256/272/302/309/322/481, 26/33/91/145/272/302/481,26/33/145/309/322/383, 26/114/145/322, 26/145/183/256/309/383,29/122/145/158/383, 29/130/145/256/272/371/394, 29/145/183/302/309/412,29/309/394, 33/122/145/272/309/412, 91/130/145/158/183/383, 91/309/383,130/145/256/272/371, 130/145/302/309, 130/272/481, 145/302/309, 268,309, 349, 360, 380, 394, 463, and 483, wherein the amino acid positionsof the polypeptide sequence are numbered with reference to SEQ ID NO:276 or 1224. In some further embodiments, the recombinant amylasecomprises a polypeptide sequence having at least 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NO: 276 or 1224, or a functional fragmentthereof, and wherein the recombinant amylase comprises at least onesubstitution or substitution set selected from16E/26E/29D/128I/145G/256A/309S/383Q,16E/26E/33D/114A/145G/309S/383Q/394E,16E/26E/33D/128I/130E/145G/302M/309S/322F/371Y,16E/26E/33D/145G/158K/383Q/394E, 16E/26E/145G/302M,16E/29D/33D/145G/256A/302M, 18S,26E/29D/33D/130E/145G/157F/158K/183G/394E,26E/29D/33D/145G/157F/183G/256A/309S/383Q,26E/29D/122A/130E/145G/183G/256A/272D/309S/394E,26E/29D/145G/157F/256A/302M/322F/481V,26E/29D/145G/256A/272D/302M/309S/322F/481V,26E/33D/91K/145G/272D/302M/481V, 26E/33D/145G/309S/322F/383Q,26E/114A/145G/322F, 26E/145G/183G/256A/309S/383Q,29D/122A/145G/158K/383Q, 29D/130E/145G/256A/272D/371Y/394E,29D/145G/183G/302M/309S/412L, 29D/309S/394E,33D/122A/145G/272D/309S/412L, 91K/130E/145G/158K/183G/383Q,91K/309S/383Q, 130E/145G/256A/272D/371Y, 130E/145G/302M/309S,130E/272D/481V, 145G/302M/309S, 268T, 309A, 349R, 360G, 380R, 394G,394N, 394S, 463M, and 483T, wherein the amino acid positions of thepolypeptide sequence are numbered with reference to SEQ ID NO: 276 or1224. In some further embodiments, the recombinant amylase comprises apolypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO: 276 or 1224, or a functional fragment thereof, and whereinthe recombinant amylase comprises at least one substitution orsubstitution set selected fromD16E/A26E/R29D/W128I/A145G/S256A/T309S/E383Q,D16E/A26E/E33D/H114A/A145G/T309S/E383Q/Q394E,D16E/A26E/E33D/W128I/D130E/A145G/Q302M/T309S/A322F/F371Y,D16E/A26E/E33D/A145G/R158K/E383Q/Q394E, D16E/A26E/A145G/Q302M,D16E/R29D/E33D/A145G/S256A/Q302M, G18S,A26E/R29D/E33D/D130E/A145G/Y157F/R158K/N183G/Q394E,A26E/R29D/E33D/A145G/Y157F/N183G/S256A/T309S/E383Q,A26E/R29D/R122A/D130E/A145G/N183G/S256A/G272D/T309S/Q394E,A26E/R29D/A145G/Y157F/S256A/Q302M/A322F/C481V,A26E/R29D/A145G/S256A/G272D/Q302M/T309S/A322F/C481V,A26E/E33D/H91K/A145G/G272D/Q302M/C481V,A26E/E33D/A145G/T309S/A322F/E383Q, A26E/H114A/A145G/A322F,A26E/A145G/N183G/S256A/T309S/E383Q, R29D/R122A/A145G/R158K/E383Q,R29D/D130E/A145G/S256A/G272D/F371Y/Q394E,R29D/A145G/N183G/Q302M/T309S/D412L, R29D/T309S/Q394E,E33D/R122A/A145G/G272D/T309S/D412L, H91K/D130E/A145G/R158K/N183G/E383Q,H91K/T309S/E383Q, D130E/A145G/S256A/G272D/F371Y,D130E/A145G/Q302M/T309S, D130E/G272D/C481V, A145G/Q302M/T309S, S268T,T309A, P349R, N360G, G380R, Q394G, Q394N, Q394S, G463M, and G483T,wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 276 or 1224.

In some additional embodiments, the recombinant amylase comprises apolypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO: 308 or 1256, or a functional fragment thereof, and whereinthe recombinant amylase comprises at least one substitution orsubstitution set at one or more positions selected from 29,37/124/250/270/349/380/394/442/491, 37/145, 37/145/250/301/349/394,37/145/258/442/491, 38/349/491, 81/259, 81/313/495, 92/257/258/338/468,92/258, 94/127/132/145/250/349, 94/270/301/349/394/442, 94/349/491,145/270, 178, 225, 226, 246, 250/349/442, 259/313/338, 270/349, 286/338,294, 313, 349, 349/380/442, 413, 413/468, 448, and 473, wherein theamino acid positions of the polypeptide sequence are numbered withreference to SEQ ID NO: 308 or 1256. In some further embodiments, therecombinant amylase comprises a polypeptide sequence having at least80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or more sequence identity to SEQ ID NO: 308 or 1256, or afunctional fragment thereof, and wherein the recombinant amylasecomprises at least one substitution or substitution set selected from29A, 37T/124N/250D/270D/349R/380R/394E/442E/491L, 37T/145G,37T/145G/250D/301K/349R/394E, 37T/145G/258S/442E/491L, 38I/349R/491L,81E/259D, 81E/313D/495L, 92E/257E/258Q/338S/468T, 92E/258Q,94G/127E/132V/145G/250D/349R, 94G/270D/301K/349R/394E/442E,94G/349R/491L, 145G/270D, 178I, 225D, 226T, 246R, 250D/349R/442E,259D/313D/338S, 270D/349R, 286T/338S, 294L, 313D, 349R, 349R/380R/442E,413Y, 413Y/468I, 448T, and 473D, wherein the amino acid positions of thepolypeptide sequence are numbered with reference to SEQ ID NO: 308 or1256. In some further embodiments, the recombinant amylase comprises apolypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO: 308 or 1256, or a functional fragment thereof, and whereinthe recombinant amylase comprises at least one substitution orsubstitution set selected from R29A,N37T/D124N/E250D/E270D/P349R/G380R/G394E/D442E/I491L, N37T/A145G,N37T/A145G/E250D/R301K/P349R/G394E, N37T/A145G/K258S/D442E/I491L,M38I/P349R/I491L, A81E/P259D, A81E/T313D/I495L,R92E/P257E/K258Q/A338S/R468T, R92E/K258Q,D94G/Q127E/I132V/A145G/E250D/P349R, D94G/E270D/R301K/P349R/G394E/D442E,D94G/P349R/I491L, A145G/E270D, V178I, E225D, Q226T, K246R,E250D/P349R/D442E, P259D/T313D/A338S, E270D/P349R, M286T/A338S, M294L,T313D, P349R, P349R/G380R/D442E, F413Y, F413Y/R468I, H448T, and E473D,wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 308 or 1256.

In some additional embodiments, the recombinant amylase comprises apolypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO: 422 or 1370, or a functional fragment thereof, and whereinthe recombinant amylase comprises at least one substitution orsubstitution set at one or more positions selected from 38,38/178/226/286/338, 38/178/286/448, 38/178/349/448, 38/226/246/258/286,38/226/258/286/338/380/448, 38/226/258/349/413, 38/226/286,38/226/286/338/380, 38/226/338/349/380, 38/226/413/448, 38/246/286,38/246/286/380, 38/258, 38/286, 38/286/413/448, 38/286/448, 38/448, 178,178/226/258/286/310/338, 178/226/286/338, 178/246/338, 178/258/286/413,178/286, 178/286/338/349, 178/286/338/448, 178/380, 178/413, 178/448,184/246/258/380, 226, 226/246/286/349/448, 226/246/413,226/258/286/349/380, 226/258/286/413/448, 226/286, 226/286/380/448,226/349/380, 246, 246/286, 246/286/380/413, 246/338, 246/413,258/286/413, 286, 286/413/448, 338/448, 349, and 413, wherein the aminoacid positions of the polypeptide sequence are numbered with referenceto SEQ ID NO: 422 or 1370. In some further embodiments, the recombinantamylase comprises a polypeptide sequence having at least 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NO: 422 or 1370, or a functional fragmentthereof, and wherein the recombinant amylase comprises at least onesubstitution or substitution set selected from 38I,38I/178I/226T/286T/338S, 38I/178I/286T/448T, 38I/178I/349R/448T,38I/226T/246R/258S/286T, 38I/226T/258S/286T/338S/380R/448T,38I/226T/258S/349R/413Y, 38I/226T/286T, 38I/226T/286T/338S/380R,38I/226T/338S/349R/380R, 38I/226T/413Y/448T, 38I/246R/286T,38I/246R/286T/380R, 38I/258S, 38I/286T, 38I/286T/413Y/448T,38I/286T/448T, 38I/448T, 178I, 178I/226T/258S/286T/310R/338S,178I/226T/286T/338S, 178I/246R/338S, 178I/258S/286T/413Y, 178I/286T,178I/286T/338S/349R, 178I/286T/338S/448T, 178I/380R, 178I/413Y,178I/448T, 184K/246R/258S/380R, 226T, 226T/246R/286T/349R/448T,226T/246R/413Y, 226T/258S/286T/349R/380R, 226T/258S/286T/413Y/448T,226T/286T, 226T/286T/380R/448T, 226T/349R/380R, 246R, 246R/286T,246R/286T/380R/413Y, 246R/338S, 246R/413Y, 246T, 258S/286T/413Y, 286T,286T/413Y/448T, 338S/448T, 349R, and 413Y, wherein the amino acidpositions of the polypeptide sequence are numbered with reference to SEQID NO: 422 or 1370. In some further embodiments, the recombinant amylasecomprises a polypeptide sequence having at least 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NO: 422 or 1370, or a functional fragmentthereof, and wherein the recombinant amylase comprises at least onesubstitution or substitution set selected from M38I,M38I/V178I/Q226T/M286T/A338S, M38I/V178I/M286T/H448T,M38I/V178I/P349R/H448T, M38I/Q226T/K246R/K258S/M286T,M38I/Q226T/K258S/M286T/A338S/G380R/H448T, M38I/Q226T/K258S/P349R/F413Y,M38I/Q226T/M286T, M38I/Q226T/M286T/A338S/G380R,M38I/Q226T/A338S/P349R/G380R, M38I/Q226T/F413Y/H448T, M38I/K246R/M286T,M38I/K246R/M286T/G380R, M38I/K258S, M38I/M286T, M38I/M286T/F413Y/H448T,M38I/M286T/H448T, M38/1H448T, V178I,V178I/Q226T/K258S/M286T/Q310R/A338S, V178I/Q226T/M286T/A338S,V178I/K246R/A338S, V178I/K258S/M286T/F413Y, V178I/M286T,V178I/M286T/A338S/P349R, V178I/M286T/A338S/H448T, V178I/G380R,V178I/F413Y, V178/1H448T, E184K/K246R/K258S/G380R, Q226T,Q226T/K246R/M286T/P349R/H448T, Q226T/K246R/F413Y,Q226T/K258S/M286T/P349R/G380R, Q226T/K258S/M286T/F413Y/H448T,Q226T/M286T, Q226T/M286T/G380R/H448T, Q226T/P349R/G380R, K246R,K246R/M286T, K246R/M286T/G380R/F413Y, K246R/A338S, K246R/F413Y, K246T,K258S/M286T/F413Y, M286T, M286T/F413Y/H448T, A338S/H448T, P349R, andF413Y, wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 422 or 1370.

In some additional embodiments, the recombinant amylase comprises apolypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO: 456 or 1404, or a functional fragment thereof, and whereinthe recombinant amylase comprises at least one substitution orsubstitution set at one or more positions selected from 29/31/92/220,29/469, 49/58/103/141/197/269/294/298, 49/58/103/194/204/298,49/58/141/298, 49/103, 103, 123, 126, 127, 131, 134, 180, 210, 214,220/469, 228, 259, 279, 282, 302, 319, 393, 415, 417, 428, 429, 466, and469, wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 456 or 1404. In some furtherembodiments, the recombinant amylase comprises a polypeptide sequencehaving at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 456 or1404, or a functional fragment thereof, and wherein the recombinantamylase comprises at least one substitution or substitution set selectedfrom 29E/31L/92A/220G, 29E/469I, 49A/58T/103L/141F/197Y/269F/294H/298Q,49A/58T/103L/194F/204D/298H, 49A/58T/141F/298H, 49A/103L, 103L, 123R,126R, 127W, 131H, 131R, 131Y, 134R, 180E, 210P, 214G, 220G/469I, 228V,259C, 279L, 282T, 302H, 302R, 302W, 319S, 393R, 393S, 415C, 417W, 428T,429S, 466C, and 469I, wherein the amino acid positions of thepolypeptide sequence are numbered with reference to SEQ ID NO: 456 or1404. In some further embodiments, the recombinant amylase comprises apolypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO: 456 or 1404, or a functional fragment thereof, and whereinthe recombinant amylase comprises at least one substitution orsubstitution set selected from R29E/F31L/R92A/A220G, R29E/V469I,S49A/S58T/V103L/Y141F/F197Y/H269F/M294H/E298Q,S49A/S58T/V103L/L194F/N204D/E298H, S49A/S58T/Y141F/E298H, S49A/V103L,V103L, D123R, T126R, Q127W, E131H, E131R, E131Y, E134R, D180E, H210P,E214G, A220G/V469I, Q228V, P259C, D279L, E282T, Q302H, Q302R, Q302W,A319S, E393R, E393S, D415C, P417W, D428T, E429S, Q466C, and V469I,wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 456 or 1404.

In some additional embodiments, the recombinant amylase comprises apolypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO: 456 or 1404, or a functional fragment thereof, and whereinthe recombinant amylase comprises at least one substitution orsubstitution set at one or more positions selected from 92, 123, 126,127, 129, 131, 134, 140, 172, 180, 210, 214, 221, 228, 253, 254, 259,279, 282, 302, 304, 305, 319, 322, 346, 359, 384, 385, 387, 393, 409,412, 415, 417, 428, 429, 441, 442, 444, 446, 451, 455, 459, 466, 467,470, 473, 474, 492, and 494, wherein the amino acid positions of thepolypeptide sequence are numbered with reference to SEQ ID NO: 456 or1404. In some further embodiments, the recombinant amylase comprises apolypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO: 456 or 1404, or a functional fragment thereof, and whereinthe recombinant amylase comprises at least one substitution orsubstitution set selected from 92G, 92Q, 123C, 123G, 123R, 126Q, 126R,1271, 127R, 127S, 127V, 127W, 129C, 129W, 131H, 131R, 131Y, 134A, 134F,134R, 140V, 172K, 172R, 180E, 180P, 180Q, 210G, 210P, 214G, 214L, 214R,221T, 221V, 228V, 253R, 254G, 259C, 259R, 259W, 279L, 279V, 282R, 282T,302G, 302H, 302R, 302V, 302W, 304T, 305Q, 305R, 305W, 319L, 319S, 322R,346G, 359G, 384M, 385L, 387G, 387V, 393R, 393S, 393W, 409A, 409R, 409W,412A, 415C, 415G, 417W, 428T, 429S, 441R, 441W, 442R, 442W, 444G, 444R,444V, 446G, 451F, 455L, 455R, 455T, 455W, 459W, 466C, 467P, 467W, 470G,470L, 470S, 473M, 474G, 492G, 492R, and 494C, wherein the amino acidpositions of the polypeptide sequence are numbered with reference to SEQID NO: 456 or 1404. In some further embodiments, the recombinant amylasecomprises a polypeptide sequence having at least 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NO: 456 or 1404, or a functional fragmentthereof, and wherein the recombinant amylase comprises at least onesubstitution or substitution set selected from R92G, R92Q, D123C, D123G,D123R, T126Q, T126R, Q1271, Q127R, Q127S, Q127V, Q127W, D129C, D129W,E131H, E131R, E131Y, E134A, E134F, E134R, R140V, D172K, D172R, D180E,D180P, D180Q, H210G, H210P, E214G, E214L, E214R, R221T, R221V, Q228V,Q253R, E254G, P259C, P259R, P259W, D279L, D279V, E282R, E282T, Q302G,Q302H, Q302R, Q302V, Q302W, R304T, D305Q, D305R, D305W, A319L, A319S,A322R, W346G, E359G, T384M, Y385L, 1387G, 1387V, E393R, E393S, E393W,V409A, V409R, V409W, D412A, D415C, D415G, P417W, D428T, E429S, D441R,D441W, D442R, D442W, E444G, E444R, E444V, T446G, E451F, N455L, N455R,N455T, N455W, R459W, Q466C, E467P, E467W, V470G, V470L, V470S, E473M,N474G, E492G, E492R, and V494C, wherein the amino acid positions of thepolypeptide sequence are numbered with reference to SEQ ID NO: 456 or1404.

In some additional embodiments, the recombinant amylase comprises apolypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO: 546 or 1494, or a functional fragment thereof, and whereinthe recombinant amylase comprises at least one substitution orsubstitution set at one or more positions selected from 123,123/126/180/214/298/302/393, 123/126/180/214/393/428,123/126/210/298/302/393, 123/126/210/428, 123/126/428, 123/210/302,126/180/210/214/298/302, 126/180/210/214/428, 126/180/298/302/393,126/210/302/428, 126/214/393, 126/298/302/393, 126/298/302/393/428, 129,129/228/253, 131/228/279, 131/282, 180/214, 210/214/302/393/428,228/282/444, 253/279/470, 253/279/492, 279, 279/470, and 441, whereinthe amino acid positions of the polypeptide sequence are numbered withreference to SEQ ID NO: 546 or 1494. In some further embodiments, therecombinant amylase comprises a polypeptide sequence having at least80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or more sequence identity to SEQ ID NO: 546 or 1494, or afunctional fragment thereof, and wherein the recombinant amylasecomprises at least one substitution or substitution set selected from123R, 123R/126R/180E/214L/298Q/302W/393R, 123R/126R/180Q/214G/393R/428T,123R/126R/210P/298Q/302W/393R, 123R/126R/210P/428T, 123R/126R/428T,123R/210P/302R, 126R/180E/210P/214G/428T, 126R/180E/298Q/302R/393R,126R/180Q/210P/214L/298Q/302R, 126R/210P/302R/428T, 126R/214G/393R,126R/298Q/302H/393R, 126R/298Q/302R/393R/428T, 129W, 129W/228V/253R,131H/228V/279V, 131Y/282T, 180Q/214L, 210P/214L/302R/393R/428T,228V/282T/444R, 253R/279L/492G, 253R/279V/470S, 279L, 279L/470S, and441R, wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 546 or 1494. In some furtherembodiments, the recombinant amylase comprises a polypeptide sequencehaving at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 546 or1494, or a functional fragment thereof, and wherein the recombinantamylase comprises at least one substitution or substitution set selectedfrom D123R, D123R/T126R/D180E/E214L/H298Q/Q302W/E393R,D123R/T126R/D180Q/E214G/E393R/D428T,D123R/T126R/H210P/H298Q/Q302W/E393R, D123R/T126R/H210P/D428T,D123R/T126R/D428T, D123R/H210P/Q302R, T126R/D180E/H210P/E214G/D428T,T126R/D180E/H298Q/Q302R/E393R, T126R/D180Q/H210P/E214L/H298Q/Q302R,T126R/H210P/Q302R/D428T, T126R/E214G/E393R, T126R/H298Q/Q302H/E393R,T126R/H298Q/Q302R/E393R/D428T, D129W, D129W/Q228V/Q253R,E131H/Q228V/D279V, E131Y/E282T, D180Q/E214L,H210P/E214L/Q302R/E393R/D428T, Q228V/E282T/E444R, Q253R/D279L/E492G,Q253R/D279V/V470S, D279L, D279L/V470S, and D441R, wherein the amino acidpositions of the polypeptide sequence are numbered with reference to SEQID NO: 546 or 1494.

In some additional embodiments, the recombinant amylase comprises apolypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO: 632 or 1580, or a functional fragment thereof, and whereinthe recombinant amylase comprises at least one substitution orsubstitution set at one or more positions selected from 2, 45/166/298,48, 48/166/170/203, 52, 58/166, 58/203, 72, 82, 114, 114/223/306/308,149, 165, 166/203, 167, 170, 184/216/306/308, 184/223/306/437, 189, 203,225, 255, 258, 275, 280, 284, 294, 298, 396, 397, 411, 431, 448, 468,482, 483, and 495, wherein the amino acid positions of the polypeptidesequence are numbered with reference to SEQ ID NO: 632 or 1580. In somefurther embodiments, the recombinant amylase comprises a polypeptidesequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ IDNO: 632 or 1580, or a functional fragment thereof, and wherein therecombinant amylase comprises at least one substitution or substitutionset selected from 2L, 45S/166L/298D, 48D, 48D/166L/170S/203S, 52R,58A/166L, 58A/203S, 72T, 82A, 114R, 114R/223L/306F/308L, 149K, 165V,166L/203S, 167Y, 170S, 184D/216L/306F/308L, 184D/223L/306F/437L, 189D,203S, 225T, 255M, 255R, 258S, 275I, 275R, 280C, 284L, 284M, 294A, 298D,298N, 396E, 396Q, 397M, 411E, 431T, 448P, 468V, 482L, 483A, 495G, and495L, wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 632 or 1580. In some furtherembodiments, the recombinant amylase comprises a polypeptide sequencehaving at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 632 or1580, or a functional fragment thereof, and wherein the recombinantamylase comprises at least one substitution or substitution set selectedfrom R2L, Y45S/I166L/H298D, A48D, A48D/I166L/D170S/E203S, Y52R,T58A/I166L, T58A/E203S, S72T, Q82A, Hi 14R, H114R/V223L/Y306F/M308L,Q149K, H165V, I166L/E203S, E167Y, D170S, E184D/I216L/Y306F/M308L,E184D/V223L/Y306F/M437L, Q189D, E203S, E225T, V255M, V255R, K258S,Q275I, Q275R, Q280C, K284L, K284M, M294A, H298D, H298N, D396E, D396Q,E397M, T411E, P431T, H448P, R468V, N482L, G483A, I495G, and I495L,wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 632 or 1580.

In some additional embodiments, the recombinant amylase comprises apolypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO: 664 or 1612, or a functional fragment thereof, and whereinthe recombinant amylase comprises at least one substitution orsubstitution set at one or more positions selected from6/39/103/324/420, 41, 58/500/501, 61, 95/98/205/227/423/458,95/98/227/423, 95/205, 95/252/285/324, 123/170/197/275/298/396/448/483,123/197, 123/197/225/294/396/448/469, 123/284/294/324/396/448,134/225/275/302, 165/225/284/298/469/483, 170/275/294/298/448, 205/206,205/206/224/315/458/480, 206/227, 206/227/315/423, 225/294/298/396, 227,392/503, 458, 499, and 500, wherein the amino acid positions of thepolypeptide sequence are numbered with reference to SEQ ID NO: 664 or1612. In some further embodiments, the recombinant amylase comprises apolypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO: 664 or 1612, or a functional fragment thereof, and whereinthe recombinant amylase comprises at least one substitution orsubstitution set selected from 6I/39I/103D/324T/420V, 41F,58S/500P/501P, 61Y, 95L/252I/285V/324T, 95V/98I/205V/227V/423V/458F,95V/98I/227V/423V, 95V/205V, 123E/170S/197Y/275I/298N/396Q/448Q/483Q,123E/197Y, 123E/197Y/225G/294A/396Q/448Q/469I,123E/284L/294A/324T/396Q/448Q, 134R/225G/275I/302W,165V/225G/284L/298N/469I/483Q, 170S/275I/294A/298N/448Q, 205V/206E,205V/206E/224L/315S/458F/480L, 206E/227V, 206E/227V/315S/423V,225G/294A/298N/396Q, 227C, 392C/503L, 458F, 458Y, 499R, and 500P,wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 664 or 1612. In some furtherembodiments, the recombinant amylase comprises a polypeptide sequencehaving at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 664 or1612, or a functional fragment thereof, and wherein the recombinantamylase comprises at least one substitution or substitution set selectedfrom L6I/V39I/V103D/A324T/I420V, L41F, T58S/H500P/H501P, L61Y,I95L/V252I/T285V/A324T, I95V/L98I/I205V/T227V/S423V/W458F,I95V/L98I/T227V/S423V, I95V/I205V,D123E/D170S/F197Y/Q275I/H298N/D396Q/H448Q/G483Q, D123E/F197Y,D123E/F197Y/E225G/M294A/D396Q/H448Q/V469I,D123E/K284L/M294A/A324T/D396Q/H448Q, E134R/E225G/Q275I/Q302W,H165V/E225G/K284L/H298N/V469I/G483Q, D170S/Q275I/M294A/H298N/H448Q,I205V/D206E, I205V/D206E/M224L/T315S/W458F/F480L, D206E/T227V,D206E/T227V/T315S/S423V, E225G/M294A/H298N/D396Q, T227C, I392C/H503L,W458F, W458Y, G499R, and H500P, wherein the amino acid positions of thepolypeptide sequence are numbered with reference to SEQ ID NO: 664 or1612.

In some additional embodiments, the recombinant amylase comprises apolypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO: 664 or 1612, or a functional fragment thereof, and whereinthe recombinant amylase comprises at least one substitution orsubstitution set at one or more positions selected from 95/252/285/324,123/197, 123/197/225/294/396/448/469, 134/225/275/302, 205/206, and205/206/224/315/458/480, wherein the amino acid positions of thepolypeptide sequence are numbered with reference to SEQ ID NO: 664 or1612. In some further embodiments, the recombinant amylase comprises apolypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO: 664 or 1612, or a functional fragment thereof, and whereinthe recombinant amylase comprises at least one substitution orsubstitution set selected from 95L/252I/285V/324T, 123E/197Y,123E/197Y/225G/294A/396Q/448Q/469I, 134R/225G/275I/302W, 205V/206E, and205V/206E/224L/315S/458F/480L, wherein the amino acid positions of thepolypeptide sequence are numbered with reference to SEQ ID NO: 664 or1612. In some further embodiments, the recombinant amylase comprises apolypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO: 664 or 1612, or a functional fragment thereof, and whereinthe recombinant amylase comprises at least one substitution orsubstitution set selected from I95L/V252I/T285V/A324T, D123E/F197Y,D123E/F197Y/E225G/M294A/D396Q/H448Q/V469I, E134R/E225G/Q275I/Q302W,I205V/D206E, and I205V/D206E/M224L/T315S/W458F/F480L, wherein the aminoacid positions of the polypeptide sequence are numbered with referenceto SEQ ID NO: 664 or 1612.

In some additional embodiments, the recombinant amylase comprises apolypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO: 780 or 1728, or a functional fragment thereof, and whereinthe recombinant amylase comprises a substitution at position 126,wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 780 or 1728. In some furtherembodiments, the recombinant amylase comprises a polypeptide sequencehaving at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 780 or1728, or a functional fragment thereof, and wherein the recombinantamylase comprises at least one substitution or substitution set selectedfrom 126C, 126D, 126E, 126K, 126L, 126M, 126N, 126Q, 126S, 126T, and126W, wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 780 or 1728. In some furtherembodiments, the recombinant amylase comprises a polypeptide sequencehaving at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 780 or1728, or a functional fragment thereof, and wherein the recombinantamylase comprises at least one substitution or substitution set selectedfrom R126C, R126D, R126E, R126K, R126L, R126M, R126N, R126Q, R126S,R126T, and R126W, wherein the amino acid positions of the polypeptidesequence are numbered with reference to SEQ ID NO: 780 or 1728.

In some additional embodiments, the recombinant amylase comprises atleast one mutation in at least one position as provided in Table 4-1,4-2, 4-3, 4-4, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 4-11, 4-12, 4-13, and/or4-14, wherein the positions are numbered with reference to SEQ ID NO: 2,18, 40, 156, 174, 276, 308, 422, 456, 546, 632, 780, 988, 1104, 1122,1224, 1256, 1370, 1404, 1494, 1580, 1612, and/or 1728. In some furtherembodiments, the recombinant amylase comprises a polypeptide sequencehaving at least about 80%, 85%, about 86%, about 87%, about 88%, about89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%,about 96%, about 97%, about 98%, about 99%, or more sequence identity toat least one sequence set forth in the even-numbered sequences of SEQ IDNOS: 4-18, 22-966, and 970-1914. In some further embodiments, therecombinant amylase comprises a polypeptide sequence having at least80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or more sequence identity to at least one sequence set forthin the even-numbered sequences of SEQ ID NOS: 4-18, 22-966, and970-1914. In some embodiments, the recombinant amylase comprises apolypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto a polypeptide sequence of SEQ ID NO: 40, 156, 174, 276, 308, 422,456, 546, 632, 664, 780, 810, 988, 1104, 1122, 1224, 1256, 1370, 1404,1494, 1580, 1612, 1728, or 1758.

In some additional embodiments, the recombinant amylase comprises atleast one sequence set forth in the even-numbered sequences of SEQ IDNOS: 4-18, 22-966, and 970-1914. In some embodiments, the recombinantamylase comprises a polypeptide sequence of SEQ ID NO: 40, 156, 174,276, 308, 422, 456, 546, 632, 664, 780, 810, 988, 1104, 1122, 1224,1256, 1370, 1404, 1494, 1580, 1612, 1728, or 1758.

In some additional embodiments, the recombinant amylase retains moreenzymatic activity after exposure to high and/or low temperatures, ascompared to a reference sequence. In some embodiments, the referencesequence is wild-type amylase, while in some other embodiments, thereference sequence is another recombinant amylase. In some additionalembodiments, the recombinant amylase is more thermostable than theamylase of SEQ ID NO: 2. In some further embodiments, the recombinantamylase is more thermostable than the amylase of SEQ ID NO: 18, 40, 156,174, 276, 308, 422, 456, 546, 632, 664, 780, 988, 1104, 1122, 1224,1256, 1370, 1404, 1494, 1580, 1612, and/or 1728. In some embodiments,the recombinant amylase is stable at 30° C., 37° C., 40° C., 50° C., 60°C., and/or 95° C., and/or 40° C. to 60° C. In some embodiments, therecombinant amylase is stable at 25° C., 37° C., 42° C., and/or 48° C.In some additional embodiments, the recombinant amylase is more stablethan a reference sequence at 30° C., 37° C., 40° C., 50° C., 60° C.,and/or 95° C., and/or 40° C. to 60° C. In some embodiments, therecombinant amylase is more stable than a reference sequence at 30° C.,37° C., 40° C., 50° C., 60° C., and/or 95° C., and/or 40° C. to 60° C.In some additional embodiments, the recombinant amylase is morethermostable than the amylase of SEQ ID NO: 2. In some furtherembodiments, the recombinant amylase is more thermostable than theamylase of SEQ ID NO: 18, 40, 156, 174, 276, 308, 422, 456, 546, 632,664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494, 1580, 1612,and/or 1728.

In some embodiments, the recombinant amylase is stable in low pHenvironments, while in other embodiments, the recombinant amylase isstable in high pH environments, and in still further embodiments, therecombinant is stable in neutral pH environments. In some embodiments,the recombinant amylase is stable in low, high, and/or neutral pHenvironments. In some embodiments, the recombinant amylase retainsenzymatic activity after exposure to a low, high, and/or neutral pHenvironment. In some additional embodiments, the recombinant amylase ismore stable in high, low, and/or neutral pH environment(s), as comparedto a reference sequence. In some embodiments, the reference sequence iswild-type amylase, while in other embodiments, the reference sequence isanother engineered amylase. In some additional embodiments, therecombinant amylase is more stable than the amylase of SEQ ID NO: 2. Insome further embodiments, the recombinant amylase is more stable thanthe amylase of SEQ ID NO: 18, 40, 156, 174, 276, 308, 422, 456, 546,632, 664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494, 1580,1612, and/or 1728. In some additional embodiments, the recombinantamylase is more stable at pHs greater than 7, than the amylase of SEQ IDNO: 2. In some additional embodiments, the recombinant amylase is morestable at pHs greater than 7, than the amylase of SEQ ID NO: 18, 40,156, 174, 276, 308, 422, 456, 546, 632, 664, 780, 988, 1104, 1122, 1224,1256, 1370, 1404, 1494, 1580, 1612, and/or 1728. In some additionalembodiments, the recombinant amylase is more stable at pH 7.8, than theamylase of SEQ ID NO: 2. In some additional embodiments, the recombinantamylase is more stable at pH 7.8, than the amylase of SEQ ID NO: 18, 40,156, 174, 276, 308, 422, 456, 546, 632, 664, 780, 988, 1104, 1122, 1224,1256, 1370, 1404, 1494, 1580, 1612, and/or 1728. In some additionalembodiments, the recombinant amylase is more stable at pH 7.5, than theamylase of SEQ ID NO: 2. In some additional embodiments, the recombinantamylase is more stable at pH 7.5, than the amylase of SEQ ID NO: 18, 40,156, 174, 276, 308, 422, 456, 546, 632, 664, 780, 988, 1104, 1122, 1224,1256, 1370, 1404, 1494, 1580, 1612, and/or 1728. In some additionalembodiments, the recombinant amylase is more stable at pH 7, than theamylase of SEQ ID NO: 2. In some additional embodiments, the recombinantamylase is more stable at pH 7, than the amylase of SEQ ID NO: 18, 40,156, 174, 276, 308, 422, 456, 546, 632, 664, 780, 988, 1104, 1122, 1224,1256, 1370, 1404, 1494, 1580, 1612, and/or 1728. In some furtherembodiments, the recombinant amylase is more stable at pHs less than 7(i.e., under acidic pH conditions or levels), than the amylase of SEQ IDNO: 2. In some additional embodiments, the recombinant amylase is morestable at pHs less than pH 7 (i.e., under acidic pH conditions orlevels), than the amylase of SEQ ID NO: 18, 40, 156, 174, 276, 308, 422,456, 546, 632, 664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494,1580, 1612, and/or 1728. In some additional embodiments, the recombinantamylase is more stable at pH 6.8, than the amylase of SEQ ID NO: 2. Insome additional embodiments, the recombinant amylase is more stable atpH 6.8, than the amylase of SEQ ID NO: 18, 40, 156, 174, 276, 308, 422,456, 546, 632, 664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494,1580, 1612, and/or 1728. In some additional embodiments, the recombinantamylase is more stable at pH 6.7, than the amylase of SEQ ID NO: 2. Insome additional embodiments, the recombinant amylase is more stable atpH 6.7, than the amylase of SEQ ID NO: 18, 40, 156, 174, 276, 308, 422,456, 546, 632, 664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494,1580, 1612, and/or 1728. In some additional embodiments, the recombinantamylase is more stable at pH 6.5, than the amylase of SEQ ID NO: 2. Insome additional embodiments, the recombinant amylase is more stable atpH 6.5, than the amylase of SEQ ID NO: 18, 40, 156, 174, 276, 308, 422,456, 546, 632, 664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494,1580, 1612, and/or 1728. In some additional embodiments, the recombinantamylase is more stable at pH 6, than the amylase of SEQ ID NO: 2. Insome additional embodiments, the recombinant amylase is more stable atpH 6, than the amylase of SEQ ID NO: 18, 40, 156, 174, 276, 308, 422,456, 546, 632, 664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494,1580, 1612, and/or 1728. In some further embodiments, the recombinantamylase is more stable at pHs less than 5, than the amylase of SEQ IDNO: 2. In some additional embodiments, the recombinant amylase is morestable at pH 5, than the amylase of SEQ ID NO: 2. In some additionalembodiments, the recombinant amylase is more stable at pH 5, than theamylase of SEQ ID NO: 18, 40, 156, 174, 276, 308, 422, 456, 546, 632,664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494, 1580, 1612,and/or 1728. In some further embodiments, the recombinant amylase ismore stable at pH 4, than the amylase of SEQ ID NO: 2. In some furtherembodiments, the recombinant amylase is more stable at pH 5, than theamylase of SEQ ID NO: 18, 40, 156, 174, 276, 308, 422, 456, 546, 632,664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494, 1580, 1612,and/or 1728. In some additional embodiments, the recombinant amylase ismore stable at pH 3.8, than the amylase of SEQ ID NO: 2. In someadditional embodiments, the recombinant amylase is more stable at pH3.8, than the amylase of SEQ ID NO: 18, 40, 156, 174, 276, 308, 422,456, 546, 632, 664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494,1580, 1612, and/or 1728.

In some additional embodiments, the recombinant amylase is more stableat pH 3.5, than the amylase of SEQ ID NO: 2. In some additionalembodiments, the recombinant amylase is more stable at pH 3.5, than theamylase of SEQ ID NO: 18, 40, 156, 174, 276, 308, 422, 456, 546, 632,664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494, 1580, 1612,and/or 1728. In some additional embodiments, the recombinant amylase ismore stable at pH 3.25, than the amylase of SEQ ID NO: 2. In someadditional embodiments, the recombinant amylase is more stable at pH3.25, than the amylase of SEQ ID NO: 18, 40, 156, 174, 276, 308, 422,456, 546, 632, 664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494,1580, 1612, and/or 1728. In some further embodiments, the recombinantamylase is more stable at pH 3, than the amylase of SEQ ID NO: 2. Insome further embodiments, the recombinant amylase is more stable at pH3, than the amylase of SEQ ID NO: 18, 40, 156, 174, 276, 308, 422, 456,546, 632, 664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494, 1580,1612, and/or 1728. In some additional embodiments, the recombinantamylase is more stable at pH 2.75, than the amylase of SEQ ID NO: 2. Insome additional embodiments, the recombinant amylase is more stable atpH 2.75, than the amylase of SEQ ID NO: 18, 40, 156, 174, 276, 308, 422,456, 546, 632, 664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494,1580, 1612, and/or 1728. In some further embodiments, the recombinantamylase is more stable at pH 2.5, than the amylase of SEQ ID NO: 2. Insome further embodiments, the recombinant amylase is more stable at pH2.5, than the amylase of SEQ ID NO: 18, 40, 156, 174, 276, 308, 422,456, 546, 632, 664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494,1580, 1612, and/or 1728. In some further embodiments, the recombinantamylase is more stable at pH 2.3, than the amylase of SEQ ID NO: 2. Insome further embodiments, the recombinant amylase is more stable at pH2.3, than the amylase of SEQ ID NO: 18, 40, 156, 174, 276, 308, 422,456, 546, 632, 664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494,1580, 1612, and/or 1728. In some further embodiments, the recombinantamylase is more stable at pH 2, than the amylase of SEQ ID NO: 2. Insome further embodiments, the recombinant amylase is more stable at pH2, than the amylase of SEQ ID NO: 18, 40, 156, 174, 276, 308, 422, 456,546, 632, 664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494, 1580,1612, and/or 1728.

In some embodiments, the recombinant amylase is more resistant toproteolysis than the amylase of SEQ ID NO: 2. In some embodiments, therecombinant amylase is more resistant to proteolysis than the amylase ofSEQ ID NO: 18, 40, 156, 174, 276, 308, 422, 456, 546, 632, 664, 780,988, 1104, 1122, 1224, 1256, 1370, 1404, 1494, 1580, 1612, and/or 1728.In some embodiments, the recombinant amylase is resistant to proteolysisby pepsin. In some additional embodiments, the recombinant amylase ismore resistant to proteolysis by pepsin than the amylase of SEQ ID NO:2. In some embodiments, the recombinant amylase is more resistant toproteolysis by pepsin than the amylase of SEQ ID NO: 18, 40, 156, 174,276, 308, 422, 456, 546, 632, 664, 780, 988, 1104, 1122, 1224, 1256,1370, 1404, 1494, 1580, 1612, and/or 1728.

In some additional embodiments, the recombinant amylase is active in thepresence of at least one bile salt. In some additional embodiments, therecombinant amylase is more active in the presence of at least one bilesalt than the amylase of SEQ ID NO: 2. In some additional embodiments,the recombinant amylase is more active in the presence of at least onebile salt than the amylase of SEQ ID NO: 18, 40, 156, 174, 276, 308,422, 456, 546, 632, 664, and/or 780, 988, 1104, 1122, 1224, 1256, 1370,1404, 1494, 1580, 1612, and/or 1728. In some embodiments, therecombinant amylase retains enzymatic activity after exposure to a bilesalt. In some additional embodiments, the recombinant amylase retainsmore enzymatic activity after exposure to a bile salt, as compared to areference sequence. In some embodiments, the reference sequence is theamylase of SEQ ID NO: 2, 18, 40, 156, 174, 276, 308, 422, 456, 546, 632,664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494, 1580, 1612,and/or 1728. In some additional embodiments, the bile salt istaurocholate.

In yet some additional embodiments, the recombinant amylase exhibitsmore than one improved property, as compared to wild-type amylase oranother recombinant amylase. In some embodiments, the recombinantamylase exhibits more than one improved property as compared to SEQ IDNO:2, while in some additional embodiments, the recombinant amylaseexhibits more than one improved property as compared to SEQ ID NO: 18,40, 156, 174, 276, 308, 422, 456, 546, 632, 664, 780, 988, 1104, 1122,1224, 1256, 1370, 1404, 1494, 1580, 1612, and/or 1728. In someembodiments, the improved properties are selected from acid stability,alkaline stability, stability and/or activity at acidic pH, stability atalkaline pH, stability at neutral pH, thermostability, proteolysisresistance, autolysis resistance, and increased activity in the presenceof at least one bile salt. In yet some additional embodiments, therecombinant amylase is more stable and/or active at acidic pHs, morethermostable, more resistant to proteolysis, and/or more active in thepresence of at least one bile salt than the amylase of SEQ ID NO: 2, 18,40, 156, 174, 276, 308, 422, 456, 546, 632, 664, 780, 988, 1104, 1122,1224, 1256, 1370, 1404, 1494, 1580, 1612, and/or 1728. It iscontemplated that any combination of improved properties will find usein the present invention. It is not intended that the present inventionbe limited to any specific combination of improved properties.Furthermore, in some embodiments, there are two improved properties,while in some other embodiments, there are three improved properties, insome additional embodiments, there are four improved properties, and insome additional embodiments, there are five or more improved properties.It is also contemplated that the recombinant amylase of the presentinvention furthers comprise additional improvements. In someembodiments, these additional improvements provide advantages overwild-type amylase, while in some other embodiments, the additionalimprovements will provide advantages over other recombinant amylases.

In some embodiments, the recombinant amylase exhibits at least oneimproved property selected from improved stability and/or activity atacidic pHs, improved thermostability, improved resistance toproteolysis, and/or improved activity in the presence of at least onebile salt, as compared to the amylase of SEQ ID NO: 2. In someembodiments, the recombinant amylase exhibits at least one improvedproperty selected from improved stability and/or activity at acidic pHs,improved thermostability, improved resistance to proteolysis, and/orimproved activity in the presence of at least one bile salt, as comparedto the amylase of SEQ ID NO: 18, 40, 156, 174, 276, 308, 422, 456, 546,632, 664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494, 1580,1612, and/or 1728. In some embodiments, the recombinant amylase exhibitsat least two improved properties selected from improved stability and/oractivity at acidic pHs, improved thermostability, improved resistance toproteolysis, and/or improved activity in the presence of at least onebile salt, as compared to the amylase of SEQ ID NO: 2. In someembodiments, the recombinant amylase exhibits at least two improvedproperties selected from improved stability and/or activity at acidicpHs, improved thermostability, improved resistance to proteolysis,and/or improved activity in the presence of at least one bile salt, ascompared to the amylase of SEQ ID NO: 18, 40, 156, 174, 276, 308, 422,456, 546, 632, 664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494,1580, 1612, and/or 1728. In some embodiments, the recombinant amylaseexhibits at least three improved properties selected from improvedstability and/or activity at acidic pHs, improved thermostability,improved resistance to proteolysis, and/or improved activity in thepresence of at least one bile salt, as compared to the amylase of SEQ IDNO: 2. In some embodiments, the recombinant amylase exhibits at leastthree improved properties selected from improved stability and/oractivity at acidic pHs, improved thermostability, improved resistance toproteolysis, and/or improved activity in the presence of at least onebile salt, as compared to the amylase of SEQ ID NO: 18, 40, 156, 174,276, 308, 422, 456, 546, 632, 664, 780, 988, 1104, 1122, 1224, 1256,1370, 1404, 1494, 1580, 1612, and/or 1728. In some embodiments, therecombinant amylase exhibits the improved properties of improvedstability and/or activity at acidic pHs, improved thermostability,improved resistance to proteolysis, and improved activity in thepresence of at least one bile salt, as compared to the amylase of SEQ IDNO: 2. In some embodiments, the recombinant amylase exhibits theimproved properties of improved stability and/or activity at acidic pHs,improved thermostability, improved resistance to proteolysis, and/orimproved activity in the presence of at least one bile salt, as comparedto the amylase of SEQ ID NO: 18, 40, 156, 174, 276, 308, 422, 456, 546,632, 664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494, 1580,1612, and/or 1728. In some embodiments, the recombinant amylase exhibitsat least one improved property selected from improved stability and/oractivity at acidic pHs, improved thermostability, improved resistance toproteolysis, and/or improved activity in the presence of at least onebile salt, as well as at least one additional improved property, ascompared to the amylase of SEQ ID NO: 2. In some embodiments, therecombinant amylase exhibits at least one improved property selectedfrom improved stability and/or activity at acidic pHs, improvedthermostability, improved resistance to proteolysis, and/or improvedactivity in the presence of at least one bile salt, as well as at leastone additional improved property, as compared to the amylase of SEQ IDNO: 18, 40, 156, 174, 276, 308, 422, 456, 546, 632, 664, 780, 988, 1104,1122, 1224, 1256, 1370, 1404, 1494, 1580, 1612, and/or 1728. In someembodiments, the recombinant amylase exhibits at least one improvedproperty selected from: i) enhanced catalytic activity; ii) increasedtolerance to acid pH; iii) increased tolerance to pH 2.3, 2.5, 2.75, 3,3.25, and/or 3.5; iv) increased tolerance to pH 6.7 and/or 6.8; v)increased tolerance to at least one protease; vi) increased tolerance toat least one bile salt; vii) increased thermotolerance; or a combinationof any of i), ii), iii), iv), v), vi), and vii) as compared to areference sequence. In some embodiments, the reference sequence is SEQID NO: 2, while in some alternative embodiments, the reference sequenceis selected from SEQ ID NO: 18, 40, 156, 174, 276, 308, 422, 456, 546,632, 664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494, 1580,1612, and/or 1728. In some embodiments, the recombinant amylase exhibitsat least one improved property selected from: i) enhanced catalyticactivity; ii) increased tolerance to acid pH; iii) increased toleranceto pH 2.3, 2.5, 2.75, 3, 3.25, and/or 3.5; iv) increased tolerance to pH6.7 and/or 6.8; v) increased tolerance to at least one protease; vi)increased tolerance to at least one bile salt; vii) increasedthermotolerance; or a combination of any of i), ii), iii), iv), v), vi),and vii) as compared to at least one reference sequence. In someembodiments, the reference sequence is SEQ ID NO: 2, while in somealternative embodiments, the reference sequence is selected from SEQ IDNO: 18, 40, 156, 174, 276, 308, 422, 456, 546, 632, 664, 780, 988, 1104,1122, 1224, 1256, 1370, 1404, 1494, 1580, 1612, and/or 1728. In someembodiments, the recombinant amylase exhibits at least one improvedproperty selected from: i) enhanced catalytic activity; ii) increasedtolerance to acid pH; iii) increased tolerance to pH 2.3, 2.5, 2.75, 3,3.25, and/or 3.5; iv) increased tolerance to pH 6.7 and/or 6.8; v)increased tolerance to at least one protease; vi) increased tolerance toat least one bile salt; vii) increased thermotolerance; or a combinationof any of i), ii), iii), iv), v), vi), and vii) as compared to at leasttwo or more reference sequences. In some embodiments, the referencesequence is selected from SEQ ID NO: 18, 40, 156, 174, 276, 308, 422,456, 546, 632, 664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494,1580, 1612, and/or 1728. In some embodiments, the protease is selectedfrom pepsin, trypsin, and/or chymotrypsin. In some embodiments, theincreased thermotolerance is measured at 30° C., 37° C., 40° C., 50° C.,60° C., and/or 95° C., and/or 40° C. to 60° C. In some furtherembodiments, the recombinant amylase is purified.

In still some further embodiments, the amylase is stable in food and/orbeverages. In some additional embodiments, the amylase is stable innutritional and other supplements. In some embodiments, the supplementsare liquid, while in other embodiments, they are emulsions, suspensions,or solids. It is not intended that the present invention be limited toany particular food, beverage, and/or supplement format or form.

The present invention also provides recombinant polynucleotide sequencesencoding at least one recombinant amylase provided herein. In someembodiments, the recombinant polynucleotide sequence is codon-optimized.In some further embodiments, the recombinant polynucleotide comprises asequence having at least about 85%, about 86%, about 87%, about 88%,about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about95%, about 96%, about 97%, about 98%, about 99%, or more sequenceidentity to at least one sequence set forth in the odd-numberedsequences of SEQ ID NOS: 3-17, 21-965, and 969-1913. In some furtherembodiments, the polynucleotide comprises a sequence having at least85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or more sequence identity to at least one sequence set forth in theodd-numbered sequences of SEQ ID NOS: 3-17, 21-965, and 969-1913. Insome additional embodiments, the present invention provides at least onesequence set forth in the odd-numbered sequences of SEQ ID NOS: 3-17,21-965, and 969-1913. In some additional embodiments, the recombinantpolynucleotide sequence comprises a sequence selected from theodd-numbered sequences of SEQ ID NOS: 3-17, 21-965, and 969-1913. Insome embodiments, the recombinant polynucleotide sequence comprises asequence selected from the odd-numbered sequences of SEQ ID NOS: 3-17,21-965, and 969-1913, wherein said sequence encodes a recombinantpolypeptide provided herein. In some embodiments, the recombinantpolynucleotide sequence comprises a sequence selected from theodd-numbered sequences of SEQ ID NOS: 3-17, 21-965, and 969-1913,wherein said sequence encodes a recombinant polypeptide provided in aneven-numbered sequence of SEQ ID NOS: 4-18, 22-966, and 970-1914. Insome further embodiments, the recombinant polynucleotide encoding arecombinant amylase provided herein comprises a sequence having at leastabout 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98%, about 99%, or more sequence identity to at least one sequenceset forth in the odd-numbered sequences of SEQ ID NOS: 3-17, 21-965, and969-1913. In some further embodiments, the recombinant polynucleotideencoding a recombinant amylase provided herein comprises a sequencehaving at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more sequence identity to at least one sequenceset forth in the odd-numbered sequences of SEQ ID NOS: 3-17, 21-965, and969-1913. In some further embodiments, the recombinant polynucleotideencoding a recombinant amylase provided herein comprises a sequencehaving at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more sequence identity to at least one sequenceset forth in the odd-numbered sequences of SEQ ID NOS: 3-17, 21-965, and969-1913, wherein the recombinant amylase comprises a polypeptidesequence comprising an even-numbered sequence provided in SEQ ID NOS:4-18, 22-966, and 970-1914.

The present invention also provides expression vectors comprising atleast one recombinant polynucleotide sequence provided herein. Thepresent invention also provides expression vectors comprising at leastone recombinant polynucleotide sequence encoding at least onerecombinant amylase provided herein. In some additional embodiments, therecombinant polynucleotide sequence is operably linked to a controlsequence. In some embodiments, the control sequence is a promoter. Insome further embodiments, the promoter is a heterologous promoter.

The present invention also provides host cells comprising at least oneexpression vector provided herein. The present invention also provideshost cells comprising at least one expression vector comprising at leastone recombinant polynucleotide sequence encoding at least onerecombinant amylase provided herein. The present invention also provideshost cells comprising an expression vector comprising at least onerecombinant polynucleotide sequence encoding at least one recombinantamylase provided herein. The present invention also provides host cellscomprising an expression vector comprising at least one recombinantpolynucleotide sequence encoding a recombinant amylase provided herein.In some embodiments, the host cell is eukaryotic, while in somealternative embodiments, the host cell is prokaryotic. In someembodiments, the host cell is Escherichia coli. In some alternativeembodiments, the host cell is Saccharomyces cerevisiae.

The present invention also provides methods of producing at least onerecombinant amylase, comprising culturing at least one host cellprovided herein, under conditions that the recombinant amylase encodedby the recombinant polynucleotide is produced. In some embodiments, themethods further comprise the step of recovering the amylase. In yet someadditional embodiments, the methods further comprise the step ofpurifying the amylase.

The present invention also provides compositions comprising at least onerecombinant amylase provided herein. In some embodiments, thecomposition comprising at least one recombinant amylase comprises apharmaceutical composition. In some additional embodiments, thepharmaceutical composition is suitable for the treatment of pancreaticinsufficiency. In some additional embodiments, the pharmaceuticalcomposition further comprising a pharmaceutically acceptable carrierand/or excipient. In some embodiments, the pharmaceutical composition issuitable for parenteral injection or infusion, subcutaneous injection,inhalation, and/or dermal application to a human. In some embodiments,the pharmaceutical composition is suitable for oral administration to ahuman. In some embodiments, the pharmaceutical composition furthercomprises an enteric coating.

In some additional embodiments, the pharmaceutical composition furthercomprises at least one lipase. In some further embodiments, thepharmaceutical composition further comprises at least one protease. Inyet some additional embodiments, the pharmaceutical composition furthercomprises at least one protease and at least one lipase. In someembodiments, the pharmaceutical composition further comprises apancreatic extract comprising lipase, protease, and amylase activity.The present invention also provides compositions comprising at least onerecombinant amylase provided herein, wherein the compositions aresuitable for other uses.

The present invention also provides methods for treating and/orpreventing the symptoms of pancreatic insufficiency in a subject,comprising providing a subject having pancreatic insufficiency, andproviding the pharmaceutical composition provided herein to the subject.In some embodiments, the symptoms of pancreatic insufficiency areameliorated in a subject, upon the administration of the compositioncomprising at least one recombinant amylase to the subject. In somefurther embodiments, the subject is able to eat a diet that is lessrestricted in its carbohydrate content (e.g., starches) than dietsrequired by subjects exhibiting the symptoms of pancreaticinsufficiency. In some additional embodiments, the subject is a humanpatient. In some embodiments, the human patient is an infant, while insome other embodiments, the human patient is a child. In yet somefurther embodiments, the human patient is an adult, while in somealternative embodiments, the human patient is a young adult. The someembodiments, the subject is a non-human mammal, such as a non-humanprimate, pigs, cows, dogs, and cats. The present invention furtherprovides medicaments comprising at least one recombinant amylaseprovided herein. The present invention also provides for use of thecompositions comprising at least one recombinant amylase providedherein.

The present invention further provides methods for starch hydrolysis,comprising providing starch and at least one engineered amylase providedherein; and exposing the starch to the engineered amylase underconditions such that the starch is hydrolyzed by said engineeredamylase. In some embodiments, the present invention provides methods forstarch hydrolysis comprising providing starch and a compositioncomprising at least one engineered amylase, and exposing said starch tosaid composition under conditions such that said starch is hydrolyzed bysaid engineered amylase of said composition.

DESCRIPTION OF THE INVENTION

The present invention provides engineered amylase polypeptides andcompositions thereof. The engineered amylase polypeptides have beenoptimized to provide improved thermostability, protease stability, andstability under a range of pH conditions, including acidic (pH<7)conditions. The invention also relates to the use of the compositionscomprising the engineered amylase polypeptides for therapeutic and/ornutritional purposes. In some embodiments, the amylase variants of thepresent invention find use in PERT treatment of PEI conditions. In someadditional embodiments, the amylase is administered in formats that donot require an enteric coating and/or proton-pump inhibitors (PPIs). Thepresent invention also provides polynucleotides encoding the engineeredamylase polypeptides, as well as methods for making the engineeredpolynucleotides and amylase polypeptides.

Abbreviations and Definitions

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention pertains. Generally,the nomenclature used herein and the laboratory procedures of cellculture, molecular genetics, microbiology, biochemistry, organicchemistry, analytical chemistry and nucleic acid chemistry describedbelow are those well-known and commonly employed in the art. Suchtechniques are well-known and described in numerous texts and referenceworks well known to those of skill in the art. Standard techniques, ormodifications thereof, are used for chemical syntheses and chemicalanalyses. All patents, patent applications, articles and publicationsmentioned herein, both supra and infra, are hereby expresslyincorporated herein by reference.

Although any suitable methods and materials similar or equivalent tothose described herein find use in the practice of the presentinvention, some methods and materials are described herein. It is to beunderstood that this invention is not limited to the particularmethodology, protocols, and reagents described, as these may vary,depending upon the context they are used by those of skill in the art.Accordingly, the terms defined immediately below are more fullydescribed by reference to the application as a whole. All patents,patent applications, articles and publications mentioned herein, bothsupra and infra, are hereby expressly incorporated herein by reference.

As used herein, the singular “a”, “an,” and “the” include the pluralreferences, unless the context clearly indicates otherwise.

Numeric ranges are inclusive of the numbers defining the range. Thus,every numerical range disclosed herein is intended to encompass everynarrower numerical range that falls within such broader numerical range,as if such narrower numerical ranges were all expressly written herein.It is also intended that every maximum (or minimum) numerical limitationdisclosed herein includes every lower (or higher) numerical limitation,as if such lower (or higher) numerical limitations were expresslywritten herein.

As used herein, the term “about” means an acceptable error for aparticular value. In some instances, “about” means within 0.05%, 0.5%,1.0%, or 2.0%, of a given value range. In some instances, “about” meanswithin 1, 2, 3, or 4 standard deviations of a given value.

Furthermore, the headings provided herein are not limitations of thevarious aspects or embodiments of the invention which can be had byreference to the application as a whole. Accordingly, the terms definedimmediately below are more fully defined by reference to the applicationas a whole. Nonetheless, in order to facilitate understanding of theinvention, a number of terms are defined below.

Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation; amino acid sequences are written left to right inamino to carboxy orientation, respectively.

As used herein, the term “comprising” and its cognates are used in theirinclusive sense (i.e., equivalent to the term “including” and itscorresponding cognates).

As used herein, “EC” number refers to the Enzyme Nomenclature of theNomenclature Committee of the International Union of Biochemistry andMolecular Biology (NC-IUBMB). The IUBMB biochemical classification is anumerical classification system for enzymes based on the chemicalreactions they catalyze.

As used herein “ATCC” refers to the American Type Culture Collectionwhose biorepository collection includes genes and strains.

As used herein “NCBI” refers to National Center for BiologicalInformation and the sequence databases provided therein.

As used herein, “amylase” refers to an enzyme that is capable ofhydrolyzing starch to produce glucose and maltose.

As used herein, “protease” (and “proteinase” and “peptidase”) refers tothe numerous enzymes that hydrolyze proteins. There are numerousproteases involved in the breakdown of proteins into smaller polypeptideunties or single amino acids. Proteases are important for numerousbiological functions, including digestion of ingested proteins, proteincatabolism, and cell signaling.

As used herein, “lipase” refers to any enzyme commonly referred to as“lipase,” that catalyzes the hydrolysis of fats by hydrolyzing the esterbonds of triglycerides. Pancreatic lipases are important in thebreakdown of fats to fatty acids, glycerol, and other alcohols. Lipasesare essential in the digestion, transport, and processing of dietarylipids in most organisms.

As used herein, the term “lipid” refers to a class of water-insolublemacromolecules that include fatty acids and their esters, sterols,prenols, certain poorly soluble vitamins, and other related compounds.“Fats” are a subset of lipids composed of fatty acid esters (e.g.,triglycerides, which are made from glycerol and three fatty acids). Itis not intended that the present invention be limited to any specificlipid and/or fat. Taking the context into consideration, the terms “fat”and “lipid” are used interchangeably herein.

As used herein, the terms “protein,” “polypeptide,” and “peptide” areused interchangeably to denote a polymer of at least two amino acidscovalently linked by an amide bond, regardless of length orpost-translational modification (e.g., glycosylation orphosphorylation).

As used herein, the term “amino acids” are referred to herein by eithertheir commonly known three-letter symbols or by the one-letter symbolsrecommended by IUPAC-IUB Biochemical Nomenclature Commission.Nucleotides, likewise, may be referred to by their commonly acceptedsingle letter codes.

As used herein, the term “engineered,” “recombinant,” “non-naturallyoccurring,” and “variant,” when used with reference to a cell, apolynucleotide or a polypeptide refers to a material or a materialcorresponding to the natural or native form of the material that hasbeen modified in a manner that would not otherwise exist in nature or isidentical thereto but produced or derived from synthetic materialsand/or by manipulation using recombinant techniques.

Recombinant polypeptides can be produced using any suitable methodsknown the art. Genes encoding the wild-type polypeptide of interest canbe cloned in vectors, such as plasmids, and expressed in desired hosts,such as E. coli, S. cerevisiae, etc. Variants of recombinantpolypeptides can be generated by various methods known in the art.Indeed, there is a wide variety of different mutagenesis techniques wellknown to those skilled in the art. In addition, mutagenesis kits arealso available from many commercial molecular biology suppliers. Methodsare available to make specific substitutions at defined amino acids(site-directed), specific or random mutations in a localized region ofthe gene (regio-specific), or random mutagenesis over the entire gene(e.g., saturation mutagenesis). Numerous suitable methods are known tothose in the art to generate enzyme variants, including but not limitedto site-directed mutagenesis of single-stranded DNA or double-strandedDNA using PCR, cassette mutagenesis, gene synthesis, error-prone PCR,shuffling, and chemical saturation mutagenesis, or any other suitablemethod known in the art. Non-limiting examples of methods used for DNAand protein engineering are provided in the following patents: U.S. Pat.Nos. 6,117,679; 6,420,175; 6,376,246; 6,586,182; 7,747,391; 7,747,393;7,783,428; and 8,383,346. After the variants are produced, they can bescreened for any desired property (e.g., high or increased activity, orlow or reduced activity, increased thermal activity, increased thermalstability, and/or acidic pH stability, etc.). In some embodiments,“recombinant amylase polypeptides” (also referred to herein as“engineered amylase polypeptides,” “variant amylase enzymes,” and“amylase variants”) find use.

As used herein, “wild-type” and “naturally-occurring” refer to the formfound in nature. For example, a wild-type polypeptide or polynucleotidesequence is a sequence present in an organism that can be isolated froma source in nature and which has not been intentionally modified byhuman manipulation.

As used herein, “coding sequence” refers to that part of a nucleic acid(e.g., a gene) that encodes an amino acid sequence of a protein.

The term “percent (%) sequence identity” is used herein to refer tocomparisons among polynucleotides and polypeptides, and are determinedby comparing two optimally aligned sequences over a comparison window,wherein the portion of the polynucleotide or polypeptide sequence in thecomparison window may comprise additions or deletions (i.e., gaps) ascompared to the reference sequence for optimal alignment of the twosequences. The percentage may be calculated by determining the number ofpositions at which the identical nucleic acid base or amino acid residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the window of comparison and multiplying the result by 100to yield the percentage of sequence identity. Alternatively, thepercentage may be calculated by determining the number of positions atwhich either the identical nucleic acid base or amino acid residueoccurs in both sequences or a nucleic acid base or amino acid residue isaligned with a gap to yield the number of matched positions, dividingthe number of matched positions by the total number of positions in thewindow of comparison and multiplying the result by 100 to yield thepercentage of sequence identity. Those of skill in the art appreciatethat there are many established algorithms available to align twosequences. Optimal alignment of sequences for comparison can beconducted, e.g., by the local homology algorithm of Smith and Waterman(Smith and Waterman, Adv. Appl. Math., 2:482 [1981]), by the homologyalignment algorithm of Needleman and Wunsch (Needleman and Wunsch, J.Mol. Biol., 48:443 [1970), by the search for similarity method ofPearson and Lipman (Pearson and Lipman, Proc. Natl. Acad. Sci. USA85:2444 [1988]), by computerized implementations of these algorithms(e.g., GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin SoftwarePackage), or by visual inspection, as known in the art. Examples ofalgorithms that are suitable for determining percent sequence identityand sequence similarity include, but are not limited to the BLAST andBLAST 2.0 algorithms, which are described by Altschul et al. (See,Altschul et al., J. Mol. Biol., 215: 403-410 [1990]; and Altschul etal., Nucleic Acids Res., 3389-3402 [1977], respectively). Software forperforming BLAST analyses is publicly available through the NationalCenter for Biotechnology Information website. This algorithm involvesfirst identifying high scoring sequence pairs (HSPs) by identifyingshort words of length W in the query sequence, which either match orsatisfy some positive-valued threshold score T when aligned with a wordof the same length in a database sequence. T is referred to as, theneighborhood word score threshold (See, Altschul et al., supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are then extended inboth directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always >0) and N (penalty score formismatching residues; always <0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlength(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix(See, Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915[1989]). Exemplary determination of sequence alignment and % sequenceidentity can employ the BESTFIT or GAP programs in the GCG WisconsinSoftware package (Accelrys, Madison Wis.), using default parametersprovided.

As used herein, “reference sequence” refers to a defined sequence usedas a basis for a sequence comparison. A reference sequence may be asubset of a larger sequence, for example, a segment of a full-lengthgene or polypeptide sequence. Generally, a reference sequence is atleast 20 nucleotide or amino acid residues in length, at least 25residues in length, at least 50 residues in length, at least 100residues in length or the full length of the nucleic acid orpolypeptide. Since two polynucleotides or polypeptides may each (1)comprise a sequence (i.e., a portion of the complete sequence) that issimilar between the two sequences, and (2) may further comprise asequence that is divergent between the two sequences, sequencecomparisons between two (or more) polynucleotides or polypeptide aretypically performed by comparing sequences of the two polynucleotides orpolypeptides over a “comparison window” to identify and compare localregions of sequence similarity. In some embodiments, a “referencesequence” can be based on a primary amino acid sequence, where thereference sequence is a sequence that can have one or more changes inthe primary sequence. “Comparison window” refers to a conceptual segmentof at least about 20 contiguous nucleotide positions or amino acidsresidues wherein a sequence may be compared to a reference sequence ofat least 20 contiguous nucleotides or amino acids and wherein theportion of the sequence in the comparison window may comprise additionsor deletions (i.e., gaps) of 20 percent or less as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The comparison window can belonger than 20 contiguous residues, and includes, optionally 30, 40, 50,100, or longer windows.

“Corresponding to”, “reference to” or “relative to” when used in thecontext of the numbering of a given amino acid or polynucleotidesequence refers to the numbering of the residues of a specifiedreference sequence when the given amino acid or polynucleotide sequenceis compared to the reference sequence. In other words, the residuenumber or residue position of a given polymer is designated with respectto the reference sequence rather than by the actual numerical positionof the residue within the given amino acid or polynucleotide sequence.For example, a given amino acid sequence, such as that of an engineeredamylase, can be aligned to a reference sequence by introducing gaps tooptimize residue matches between the two sequences. In these cases,although the gaps are present, the numbering of the residue in the givenamino acid or polynucleotide sequence is made with respect to thereference sequence to which it has been aligned.

As used herein, “mutation” refers to any change in a polypeptide orpolynucleotide sequence. It is intended to encompass any number (i.e.,one or more) of substitutions, insertions, deletions, and/orrearrangements present in a sequence (i.e., as compared to the startingor reference sequence). Thus, mutations in sequences result in theproduction of variant polypeptides (e.g., variant or recombinantamylases), as provided herein.

As used herein, “amino acid difference” or “residue difference” refersto a difference in the amino acid residue at a position of a polypeptidesequence relative to the amino acid residue at a corresponding positionin a reference sequence. The positions of amino acid differencesgenerally are referred to herein as “Xn,” where n refers to thecorresponding position in the reference sequence upon which the residuedifference is based. For example, a “residue difference at position X92as compared to SEQ ID NO: 2” refers to a difference of the amino acidresidue at the polypeptide position corresponding to position 92 of SEQID NO: 2. Thus, if the reference polypeptide of SEQ ID NO: 2 has athreonine at position 92, then a “residue difference at position X92 ascompared to SEQ ID NO:2” means there is an amino acid residue other thanthreonine at the position of the polypeptide corresponding to position92 of SEQ ID NO: 2 (e.g., T92A). In most instances herein, the specificamino acid residue difference at a position is indicated as “XnY” where“Xn” specified the corresponding position as described above, and “Y” isthe single letter identifier of the amino acid found in the engineeredpolypeptide (i.e., the different residue than in the referencepolypeptide). In some instances (e.g., in Table 4-1, 4-2, 4-3, 4-4, 4-5,4-6, 4-7, 4-8, 4-9, 4-10, 4-11, 4-12, 4-13, and/or 4-14), the presentdisclosure also provides specific amino acid differences denoted by theconventional notation “AnB”, where A is the single letter identifier ofthe residue in the reference sequence, “n” is the number of the residueposition in the reference sequence, and B is the single letteridentifier of the residue substitution in the sequence of the engineeredpolypeptide. In some instances, a polypeptide of the present disclosurecan include one or more amino acid residue differences relative to areference sequence, which is indicated by a list of the specifiedpositions where residue differences are present relative to thereference sequence. In some embodiments, where more than one amino acidcan be used in a specific residue position of a polypeptide, the variousamino acid residues that can be used are separated by a “/” (e.g.,X446A/X446G/X446M or X446A/G/M or P446A/G/M). In some embodiments, theenzyme variants comprise more than one substitution. These substitutionsare separated by a slash for ease in reading (e.g., V408A/M439S). Thepresent application includes engineered polypeptide sequences comprisingone or more amino acid differences that include either/or bothconservative and non-conservative amino acid substitutions.

As used herein, “conservative amino acid substitution” refers to asubstitution of a residue with a different residue having a similar sidechain, and thus typically involves substitution of the amino acid in thepolypeptide with amino acids within the same or similar defined class ofamino acids. By way of example and not limitation, an amino acid with analiphatic side chain may be substituted with another aliphatic aminoacid (e.g., alanine, valine, leucine, and isoleucine); an amino acidwith hydroxyl side chain is substituted with another amino acid with ahydroxyl side chain (e.g., serine and threonine); an amino acids havingaromatic side chains is substituted with another amino acid having anaromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, andhistidine); an amino acid with a basic side chain is substituted withanother amino acid with a basis side chain (e.g., lysine and arginine);an amino acid with an acidic side chain is substituted with anotheramino acid with an acidic side chain (e.g., aspartic acid or glutamicacid); and/or a hydrophobic or hydrophilic amino acid is replaced withanother hydrophobic or hydrophilic amino acid, respectively.

As used herein, “non-conservative substitution” refers to substitutionof an amino acid in the polypeptide with an amino acid withsignificantly differing side chain properties. Non-conservativesubstitutions may use amino acids between, rather than within, thedefined groups and affects (a) the structure of the peptide backbone inthe area of the substitution (e.g., proline for glycine) (b) the chargeor hydrophobicity, or (c) the bulk of the side chain. By way of exampleand not limitation, an exemplary non-conservative substitution can be anacidic amino acid substituted with a basic or aliphatic amino acid; anaromatic amino acid substituted with a small amino acid; and ahydrophilic amino acid substituted with a hydrophobic amino acid.

As used herein, “deletion” refers to modification to the polypeptide byremoval of one or more amino acids from the reference polypeptide.Deletions can comprise removal of 1 or more amino acids, 2 or more aminoacids, 5 or more amino acids, 10 or more amino acids, 15 or more aminoacids, or 20 or more amino acids, up to 10% of the total number of aminoacids, or up to 20% of the total number of amino acids making up thereference enzyme while retaining enzymatic activity and/or retaining theimproved properties of an engineered enzyme. Deletions can be directedto the internal portions and/or terminal portions of the polypeptide. Invarious embodiments, the deletion can comprise a continuous segment orcan be discontinuous.

As used herein, “insertion” refers to modification to the polypeptide byaddition of one or more amino acids from the reference polypeptide.Insertions can be in the internal portions of the polypeptide, or to thecarboxy or amino terminus. Insertions as used herein include fusionproteins as is known in the art. The insertion can be a contiguoussegment of amino acids or separated by one or more of the amino acids inthe naturally occurring polypeptide.

As used herein, an asterisk (*) used in the context of a polynucleotidesequence indicates the presence of a stop codon within thepolynucleotide sequence. In some embodiments, the variant amylases aretruncated, as compared to the starting or reference sequence, due to thepresence of stop codons.

A “functional fragment” and a “biologically active fragment” are usedinterchangeably herein refers to a polypeptide that has anamino-terminal and/or carboxy-terminal deletion(s) and/or internaldeletions (e.g., the sequence is truncated), but where the remainingamino acid sequence is identical to the corresponding positions in thesequence to which it is being compared (e.g., a full-length engineeredamylase of the present invention) and that retains substantially all ofthe activity of the full-length polypeptide.

As used herein, “isolated polypeptide” refers to a polypeptide that issubstantially separated from other contaminants that naturally accompanyit (e.g., protein, lipids, and polynucleotides). The term embracespolypeptides that have been removed or purified from theirnaturally-occurring environment or expression system (e.g., host cell orin vitro synthesis). The recombinant amylase polypeptides may be presentwithin a cell, present in the cellular medium, or prepared in variousforms, such as lysates or isolated preparations. As such, in someembodiments, the recombinant amylase polypeptides can be an isolatedpolypeptide.

The terms “isolated” and “purified” are used to refer to a molecule(e.g., an isolated nucleic acid, polypeptide, etc.) or other componentthat is removed from at least one other component with which it isnaturally associated. The term “purified” does not require absolutepurity, rather it is intended as a relative definition.

As used herein, “substantially pure polypeptide” refers to a compositionin which the polypeptide species is the predominant species present(i.e., on a molar or weight basis it is more abundant than any otherindividual macromolecular species in the composition), and is generallya substantially purified composition when the object species comprisesat least about 50 percent of the macromolecular species present by moleor % weight. Generally, a substantially pure amylase compositioncomprises about 60% or more, about 70% or more, about 80% or more, about90% or more, about 95% or more, and about 98% or more of allmacromolecular species by mole or % weight present in the composition.In some embodiments, the object species is purified from a startingpreparation to essential homogeneity (i.e., contaminant species cannotbe detected in the composition by conventional detection methods)wherein the composition consists essentially of a single macromolecularspecies. Solvent species, small molecules (<500 Daltons), and elementalion species are not considered macromolecular species. In someembodiments, the isolated recombinant amylase polypeptides aresubstantially pure polypeptide compositions. In some embodiments,substantially pure recombinant amylase polypeptide preparations added toformulations suitable for use in the present invention (e.g.,polysaccharides, surfactants, etc.).

As used herein, an “improved enzyme property” and “improved property”refers to a property of an engineered amylase polypeptide whichcomprises an improvement in any enzyme property as compared to areference amylase polypeptide and/or as a wild-type amylase polypeptideor another engineered amylase polypeptide. Improved properties includebut are not limited to such properties as increased protein expression,increased thermoactivity, increased thermostability, increased pHactivity, increased stability, increased enzymatic activity, increasedsubstrate specificity or affinity, increased specific activity,increased resistance to substrate or end-product inhibition, increasedchemical stability, improved chemoselectivity, improved solventstability, increased tolerance to acidic or basic pH, increasedtolerance to proteolytic activity (i.e., reduced sensitivity toproteolysis), reduced aggregation, increased solubility, reducedimmunogenicity, improved post-translational modification (e.g.,glycosylation), and altered temperature profile.

As used herein, “increased enzymatic activity” or “enhanced catalyticactivity” refers to an improved property of the engineered amylasepolypeptides, that can be represented by an increase in specificactivity (e.g., product produced/time/weight protein) or an increase inpercent conversion of the substrate to the product (e.g., percentconversion of starting amount of substrate to product in a specifiedtime period using a specified amount of amylase) as compared to thereference amylase enzyme. Exemplary methods to determine enzyme activityare provided in the Examples. Any property relating to enzyme activitymay be affected, including the classical enzyme properties of K_(m),V_(max) or k_(cat), changes of which can lead to increased enzymaticactivity. Improvements in enzyme activity can be from about 1.1 fold theenzymatic activity of the corresponding wild-type enzyme, to as much as2-fold, 5-fold, 10-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold,150-fold, 200-fold or more enzymatic activity than the naturallyoccurring amylase or another engineered amylase from which the amylasepolypeptides were derived.

Amylase activity can be measured by any suitable method known in the art(e.g., standard assays, such as monitoring changes in spectrophotometricproperties of reactants or products). In some embodiments, the amount ofproducts produced can be measured by using ethylidene pNP-G7 as asubstrate and monitoring the p-nitrophenol released by monitoringabsorbance at A405. In some embodiments, starch can be used as thesubstrate, and the amount of maltose produced can be measured with thedinitrosalicylic acid assay and monitoring absorbance at 540 nm, whilein some other embodiments, the products can be measured usingalternative methods known in the art. Comparisons of enzyme activitiesare made using a defined preparation of enzyme, a defined assay under aset condition, and one or more defined substrates, as further describedin detail herein.

As used herein, the terms “protease stable” and “stability toproteolysis” refer to the ability of a protein (e.g., a recombinantamylase of the present invention) to function and withstand proteolysismediated by any proteolytic enzyme or other proteolytic compound orfactor and retain its function following treatment with the protease. Itis not intended that the term be limited to the use of any particularprotease to assess the stability of a protein. Indeed, the engineeredamylases of the present invention are stable and retain enzymaticactivity in the presence of various proteases. In some embodiments, therecombinant amylases of the present invention to function and withstandproteolysis mediated by any proteolytic enzyme or other proteolyticcompound or factor and retain its function following exposure to aprotease. In some embodiments, the engineered amylases are stable in thepresence of trypsin, chymotrypsin, and/or pepsin. However, it is notintended that the present invention be limited to any specific proteaseor any particular method of assessing proteolytic stability.

As used herein, the term “pH stability” refers to the ability of aprotein (e.g., a recombinant amylase of the present invention) tofunction after incubation at a particular pH. In some embodiments, thepresent invention provides recombinant amylases that are stable at arange of pHs, including, but not limited to the range of pH 2 to pH 7.In some embodiments, the recombinant amylases are stable at different pHranges, as indicated in the Examples provided herein. It is not intendedthat the present invention be limited to any pH stability level nor pHrange.

As used herein, the term “improved tolerance to acidic pH” means that arecombinant amylase according to the invention will have increasedstability (higher retained activity at about pH 7, 6, 5, 4 3, 2, or evenlower, after exposure to acidic pH for a specified period of time [e.g.,1 hour, up to 24 hours, etc.]) as compared to a reference amylase oranother enzyme.

“Physiological pH” as used herein means the pH range generally found ina subject's (e.g., human) blood (e.g., pH 7.2-7.4).

The term “basic pH” (e.g., used with reference to improved stability tobasic pH conditions or increased tolerance to basic pH) means a pH rangeof about 7 to 11, or in some embodiments, greater than pH 11.

The term “acidic pH” (e.g., used with reference to improved stability toacidic pH conditions or increased tolerance to acidic pH) means a pHrange that encompasses any pH values less than 7. In some embodiments,the acid pH is less than 7, while in some other embodiments, the pH isless than about 6, 5, 4, 3, 2, or lower. In some alternativeembodiments, the recombinant amylases of the present invention arestable at pH levels of 2 to 4. However, it is not intended that thepresent invention be limited to any specific pH value or range ofvalues.

As used herein, the phrase “gastric challenge” refers to the exposure ofthe recombinant amylases of the present invention to a low pHenvironment and the presence of at least one enzyme (e.g., pepsin), suchthat the recombinant amylase is exposed to the conditions that may beencountered in the stomach (e.g., the human stomach).

As used herein, the phrase “intestinal challenge” refers to the exposureof the recombinant amylases of the present invention to a neutral pHenvironment and the presence of at least one protease (e.g., intestinalproteases such as trypsin and/or chymotrypsin) and/or at least one bilesalt (e.g., sodium taurocholate), such that the recombinant lipase isexposed to the conditions that may be encountered in the intestinaltract (e.g., human intestines).

As used herein, the phrase “multiple challenges in sequence” refers tothe exposure of the recombinant amylases of the present invention to aseries of challenge conditions. For example, in some embodiments, a onehour heat challenge was followed by a one hour gastric challenge, andthen followed by a one hour intestinal challenge. It is not intendedthat the present invention be limited to any specific challenges and/orchallenge conditions or a specific sequence of challenges.

The terms “thermal stability” and “thermostability” refer to the abilityof a protein (e.g., a recombinant amylase of the present invention) tofunction at a particular temperature. In some embodiments, the termrefers to the ability of a protein to function following incubation at aparticular temperature. In some embodiments, the recombinant amylases ofthe present invention are “thermotolerant” (i.e., the enzymes maintaintheir catalytic activity at elevated temperatures). In some embodiments,the recombinant amylases resist inactivation at elevated temperaturesand in some embodiments, maintain catalytic activity at elevatedtemperatures for prolonged exposure times. These terms are usedinterchangeably herein. It is not intended that the present invention belimited to any specific temperature and/or exposure time. Such stabilitycan be measured by any method known in the art (e.g., the methodsdescribed herein). It is not intended that the present invention belimited to any specific temperature stability level nor temperaturerange. In some embodiments, thermal stability is measured followingincubation of a protein (e.g., a recombinant amylase of the presentinvention) at a particular temperature.

As used herein, the term “chemical stability” refers to the ability of aprotein (e.g., a recombinant amylase of the present invention) tofunction in the presence of a chemical that adversely affects thefunction of another protein. It is not intended that the presentinvention be limited to any specific chemical stability level nor rangeof chemical stabilities.

As used herein, the term “conversion” refers to the enzymatic conversion(or biotransformation) of a substrate(s) to the correspondingproduct(s). “Percent conversion” refers to the percent of the substratethat is converted to the product within a period of time under specifiedconditions. Thus, the “enzymatic activity” or “activity” of an amylasepolypeptide can be expressed as “percent conversion” of the substrate tothe product in a specific period of time.

As used herein, “hybridization stringency” relates to hybridizationconditions, such as washing conditions, in the hybridization of nucleicacids. Generally, hybridization reactions are performed under conditionsof lower stringency, followed by washes of varying but higherstringency. The term “moderately stringent hybridization” refers toconditions that permit target-DNA to bind a complementary nucleic acidthat has about 60% identity, preferably about 75% identity, about 85%identity to the target DNA, with greater than about 90% identity totarget-polynucleotide. Exemplary moderately stringent conditions areconditions equivalent to hybridization in 50% formamide, 5× Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.2×SSPE,0.2% SDS, at 42° C. “High stringency hybridization” refers generally toconditions that are about 10° C. or less from the thermal meltingtemperature T_(m) as determined under the solution condition for adefined polynucleotide sequence. In some embodiments, a high stringencycondition refers to conditions that permit hybridization of only thosenucleic acid sequences that form stable hybrids in 0.018M NaCl at 65° C.(i.e., if a hybrid is not stable in 0.018M NaCl at 65° C., it will notbe stable under high stringency conditions, as contemplated herein).High stringency conditions can be provided, for example, byhybridization in conditions equivalent to 50% formamide, 5× Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.1×SSPE,and 0.1% SDS at 65° C. Another high stringency condition is hybridizingin conditions equivalent to hybridizing in 5×SSC containing 0.1% (w:v)SDS at 65° C. and washing in 0.1×SSC containing 0.1% SDS at 65° C. Otherhigh stringency hybridization conditions, as well as moderatelystringent conditions, are described in the references cited above.

As used herein, a “vector” is a DNA construct for introducing a DNAsequence into a cell. In some embodiments, the vector is an expressionvector that is operably linked to a suitable control sequence capable ofeffecting the expression in a suitable host of the polypeptide encodedin the DNA sequence. In some embodiments, plasmids find use as vectors.In some embodiments, an “expression vector” has a promoter sequenceoperably linked to the DNA sequence (e.g., transgene) to driveexpression in a host cell, and in some embodiments, also comprises atranscription terminator sequence.

As used herein, “codon optimized” refers to changes in the codons of thepolynucleotide encoding a protein to those preferentially used in aparticular organism such that the encoded protein is more efficientlyexpressed in the organism of interest. Although the genetic code isdegenerate in that most amino acids are represented by several codons,called “synonyms” or “synonymous” codons, it is well known that codonusage by particular organisms is nonrandom and biased towards particularcodon triplets. This codon usage bias may be higher in reference to agiven gene, genes of common function or ancestral origin, highlyexpressed proteins versus low copy number proteins, and the aggregateprotein coding regions of an organism's genome. In some embodiments, thepolynucleotides encoding the amylase enzymes may be codon optimized foroptimal production from the host organism selected for expression.

As used herein, “control sequence” includes all components that arenecessary or advantageous for the expression of a polynucleotide and/orpolypeptide of the present application. Each control sequence may benative or foreign to the nucleic acid sequence encoding the polypeptide.Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter sequence, signalpeptide sequence, initiation sequence and transcription terminator. At aminimum, the control sequences include a promoter, and transcriptionaland translational stop signals. The control sequences may be providedwith linkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe nucleic acid sequence encoding a polypeptide.

As used herein, the term “operably linked” is defined herein as aconfiguration in which a control sequence is appropriately placed (i.e.,in a functional relationship) at a position relative to a polynucleotideof interest such that the control sequence directs or regulates theexpression of the polynucleotide and/or polypeptide of interest.

As used herein, “promoter sequence” refers to a nucleic acid sequencethat is recognized by a host cell for expression of a polynucleotide ofinterest, such as a coding sequence. The promoter sequence containstranscriptional control sequences, which mediate the expression of apolynucleotide of interest. The promoter may be any nucleic acidsequence that shows transcriptional activity in the host cell of choiceincluding mutant, truncated, and hybrid promoters, and may be obtainedfrom genes encoding extracellular or intracellular polypeptides eitherhomologous or heterologous to the host cell.

As used herein, the phrase “suitable reaction conditions” refers tothose conditions in the enzymatic conversion reaction solution (e.g.,ranges of enzyme loading, substrate loading, temperature, pH, buffers,co-solvents, etc.) under which a amylase polypeptide of the presentapplication is capable of converting a substrate to the desired productcompound, Exemplary “suitable reaction conditions” are provided in thepresent application and illustrated by the Examples.

As used herein, “loading,” such as in “compound loading” or “enzymeloading” refers to the concentration or amount of a component in areaction mixture at the start of the reaction.

As used herein, the term “substrate” used in the context of an enzymaticconversion reaction process refers to the compound or molecule acted onby the amylase polypeptide.

As used herein, the term “product” used in the context of an enzymaticconversion process refers to the compound or molecule resulting from theaction of the amylase polypeptide on a substrate.

As used herein, the term “expression” includes any step involved in theproduction of the polypeptide including, but not limited to,transcription, post-transcriptional modification, translation, andpost-translational modification. In some embodiments, the term alsoencompasses secretion of the polypeptide from a cell.

As used herein, the term “produces” refers to the production of proteinsand/or other compounds by cells. It is intended that the term encompassany step involved in the production of polypeptides including, but notlimited to, transcription, post-transcriptional modification,translation, and post-translational modification. In some embodiments,the term also encompasses secretion of the polypeptide from a cell.

As used herein, an amino acid or nucleotide sequence (e.g., a promotersequence, signal peptide, terminator sequence, etc.) is “heterologous”to another sequence with which it is operably linked if the twosequences are not associated in nature.

As used herein, the terms “host cell” and “host strain” refer tosuitable hosts for expression vectors comprising DNA provided herein(e.g., the polynucleotides encoding the amylase variants). In someembodiments, the host cells are prokaryotic or eukaryotic cells thathave been transformed or transfected with vectors constructed usingrecombinant DNA techniques as known in the art.

The term “analogue” means a polypeptide having more than 70% sequenceidentity but less than 100% sequence identity (e.g., more than 75%, 78%,80%, 83%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%sequence identity) with a reference polypeptide. In some embodiments,analogues comprise polypeptides that contain one or more non-naturallyoccurring amino acid residues including, but not limited, tohomoarginine, ornithine and norvaline, as well as naturally occurringamino acids. In some embodiments, analogues also include one or moreD-amino acid residues and non-peptide linkages between two or more aminoacid residues.

As used herein, the term “culturing” refers to the growing of apopulation of cells under any suitable conditions (e.g., using a liquid,gel or solid medium). In some embodiments, the cells are microbial cells(e.g., bacteria), while in some other embodiments, the cells aremammalian cells, insect cells, or cells obtained from another animal. Itis not intended that the present invention be limited to culturing ofany particular cells or cell types or any specific method of culturing.Indeed, it is intended that the present invention encompass any suitablecell types cultured under any suitable conditions.

As used herein, the term “therapeutic” refers to a compound thatprovides beneficial or desirable effects, including medical effects,that is administered to a subject who shows signs or symptoms ofpathology.

As used herein, the term “pharmaceutical composition” refers to acomposition suitable for pharmaceutical use in a subject (e.g., human)comprising a pharmaceutically effective amount of an engineered amylasepolypeptide encompassed by the invention and an acceptable carrier.

As used herein, the term “effective amount” means an amount sufficientto produce the desired result. One of general skill in the art maydetermine what the effective amount by using routine experimentation.

As used herein, the term “subject” encompasses animals, including butnot limited to mammals such as humans, non-human primates, livestock,companion animals, and laboratory animals (e.g., rodents andlagamorphs). It is intended that the term encompass females as well asmales.

As used herein, the term “patient” means any subject that is beingassessed for, treated for, or is experiencing disease.

As used herein, the term “infant” refers to a child in the period of thefirst month after birth to approximately one (1) year of age. As usedherein, the term “newborn” refers to child in the period from birth tothe 28th day of life. the term “premature infant” refers to an infantborn after the twentieth completed week of gestation, yet before fullterm, generally weighing ˜500 to ˜2499 grams at birth. A “very low birthweight infant” is an infant weighing less than 1500 g at birth.

As used herein, the term “child” refers to a person who has not attainedthe legal age for consent to treatment or research procedures. In someembodiments, the term refers to a person between the time of birth andadolescence.

As used herein, the term “adult” refers to a person who has attainedlegal age for the relevant jurisdiction (e.g., 18 years of age in theUnited States). In some embodiments, the term refers to any fully grown,mature organism. In some embodiments, the term “young adult” refers to aperson less than 18 years of age, but who has reached sexual maturity.

As used herein, “composition” and “formulation” encompass productscomprising at least one engineered amylase of the present invention,intended for any suitable use (e.g., pharmaceutical compositions,dietary and/or nutritional supplements, feed, etc.).

As used herein, the terms “administration” and “administering” acomposition mean providing a composition of the present invention to asubject (e.g., to a person suffering from the effects of pancreaticinsufficiency).

As used herein, the terms “carrier” when used in reference to apharmaceutical composition means any of the standard pharmaceuticalcarrier, buffers, and excipients, such as stabilizers, preservatives,and adjuvants.

As used herein, the term “pharmaceutically acceptable” means a materialthat can be administered to a subject without causing any undesirablebiological effects or interacting in a deleterious manner with any ofthe components in which it is contained and that possesses the desiredbiological activity.

As used herein, the term “excipient” refers to any pharmaceuticallyacceptable additive, carrier, diluent, adjuvant, or other ingredient,other than the active pharmaceutical ingredient (API; e.g., theengineered amylase polypeptides of the present invention). Excipientsare typically included for formulation and/or administration purposes.

As used herein, the term “therapeutically effective amount” when used inreference to symptoms of disease/condition refers to the amount and/orconcentration of a compound (e.g., engineered amylase polypeptides) thatameliorates, attenuates, or eliminates one or more symptom of adisease/condition or prevents or delays the onset of symptom(s).

As used herein, the term “therapeutically effective amount” when used inreference to a disease/condition refers to the amount and/orconcentration of a composition (e.g., engineered amylase polypeptides)that ameliorates, attenuates, or eliminates the disease/condition. Insome embodiments, the term is use in reference to the amount of acomposition that elicits the biological (e.g., medical) response by atissue, system, or animal subject that is sought by the researcher,physician, veterinarian, or other clinician.

As used herein, the terms “treating,” “treat,” and “treatment” refer tomedical care given to a subject (e.g., a human patient), includingadministration of pharmaceutical compositions, such as those providedherein. It is intended that the terms “treating,” “treat” and“treatment” encompass preventative (e.g., prophylactic), as well aspalliative treatment or care. In some embodiments, treatment is providedto prevent or ameliorate the symptoms of disease. In some embodiments,the pharmaceutical compositions of the present invention find use in thetreatment or prevention of pancreatic enzyme insufficiency disease orconditions.

Engineered Amylase Polypeptides

The present invention provides engineered amylases suitable for varioususes, including treatment of pancreatic enzyme insufficiency. In someembodiments the engineered amylase exhibits at least one improvedproperty compared to wild-type amylase (e.g., the polypeptide of SEQ IDNO: 2). In some embodiments, the engineered amylase has at least about75%, at least about 75%, at least about 80%, at least about 85%, atleast about 88%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, at least about99%, or at about 100% amino acid sequence identity with SEQ ID NO: 2,18, 40, 156, 174, 276, 308, 422, 456, 546, 632, 664, 780, 988, 1104,1122, 1224, 1256, 1370, 1404, 1494, 1580, 1612, and/or 1728, and anamino acid residue difference as compared to SEQ ID NO: 2, 18, 40, 156,174, 276, 308, 422, 456, 546, 632, 664, 780, 988, 1104, 1122, 1224,1256, 1370, 1404, 1494, 1580, 1612, and/or 1728, at one or more aminoacid positions (such as at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, ormore amino acid positions compared to SEQ ID NO: 2, 18, 40, 156, 174,276, 308, 422, 456, 546, 632, 664, 780, 988, 1104, 1122, 1224, 1256,1370, 1404, 1494, 1580, 1612, and/or 1728, or a sequence having at least70%, 75%, 80%, 85%, at least 88%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99% or greater amino acid sequence identitywith SEQ ID NO: 2, 18, 40, 156, 174, 276, 308, 422, 456, 546, 632, 664,780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494, 1580, 1612, and/or1728. In some embodiment the residue difference as compared to SEQ IDNO: 2, 18, 40, 156, 174, 276, 308, 422, 456, 546, 632, 664, 780, 988,1104, 1122, 1224, 1256, 1370, 1404, 1494, 1580, 1612, and/or 1728, atone or more positions include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, or more conservative amino acid substitutions. In someembodiments, the engineered amylase polypeptide comprises a polypeptidelisted in Table 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 4-11,4-12, 4-13, and/or 4-14; or a polypeptide listed in Table 4-1, 4-2, 4-3,4-4, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 4-11, 4-12, 4-13, wherein thepolypeptide is absent the histidine tag and preceding amino acid linker.In some embodiments, the engineered amylase polypeptide comprises SEQ IDNO: 18, 40, 156, 174, 276, 308, 422, 456, 546, 632, 664, 780, 988, 1104,1122, 1224, 1256, 1370, 1404, 1494, 1580, 1612, or 1728.

The present invention provide recombinant amylases and/or biologicallyactive recombinant amylase fragments comprising amino acid sequencescomprising at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 91%, at leastabout 92%, at least about 93%, at least about 94%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, or at leastabout 99% sequence identity to SEQ ID NO: 2. In some embodiments, therecombinant amylase and/or biologically active recombinant amylasefragment comprises a polypeptide sequence having at least about 70%,about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%,about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about94%, about 95%, about 96%, about 97%, about 98%, about 99%, or moresequence identity to SEQ ID NO: 2, 18, 40, 156, 174, 276, 308, 422, 456,546, 632, 664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494, 1580,1612, or 1728.

The present invention provide recombinant amylases and/or biologicallyactive recombinant amylase fragments comprising amino acid sequencescomprising at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 2. In some embodiments, the recombinantamylase comprises a polypeptide sequence having at least 70%, 75%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or more sequence identity to SEQ ID NO: 2, or a functional fragmentthereof, and comprises one or more substitutions relative to thesequence of SEQ ID NO:2.

In some embodiments, the recombinant amylase comprises a polypeptidesequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO: 2, or a functional fragment thereof, and wherein therecombinant amylase comprises one or more substitutions at positionsselected from 2, 6, 16, 18, 19, 24, 26, 27, 29, 31, 33, 37, 38, 39, 41,45, 48, 49, 51, 52, 58, 61, 66, 72, 73, 74, 81, 82, 91, 92, 94, 95, 98,103, 111, 112, 114, 122, 123, 124, 126, 127, 128, 129, 130, 131, 132,134, 137, 140, 141, 145, 149, 157, 158, 165, 166, 167, 170, 171, 172,178, 180, 183, 184, 189, 194, 203, 204, 205, 206, 210, 214, 216, 220,221, 223, 224, 225, 226, 227, 228, 246, 250, 252, 253, 254, 255, 256,257, 258, 259, 268, 269, 270, 272, 275, 276, 279, 280, 282, 284, 285,286, 294, 295, 298, 301, 302, 304, 305, 306, 308, 309, 310, 313, 315,317, 319, 322, 324, 331, 338S, 345, 346, 349, 359, 360, 366, 371, 374,380, 383, 384, 385, 387, 388, 392, 393, 394, 396, 397, 409, 411, 412,413, 415, 417, 420, 423, 428, 429, 431, 437L 441, 442, 444, 446, 448,451, 455, 458, 459, 463, 464, 466, 467, 468, 469, 470, 473, 474, 480,481, 482, 483, 491, 492, 493, 494, and 495, wherein the amino acidpositions of the polypeptide sequence are numbered with reference to SEQID NO: 2 or 18.

In some embodiments, the one or more substitutions are at positionsselected from 16; 26; 29; 33; 49; 58, 91; 114; 122; 126; 128; 130; 141;145; 157; 158; 180; 183; 184; 205; 206; 210; 214; 223; 246; 256; 270;272; 286; 298; 302; 306; 322; 371; 380; 383; 394; 412; 413; 428; and437. In some embodiments, the one or more substitutions are at positionsselected from 16, 26, 29, 33, 49, 91, 58, 114, 122, 128, 130, 141, 145,157, 158, 183, 246, 256, 270, 272, 286, 298, 302, 322, 371, 380, 383,394, 412, and 413. In some embodiments, the one or more substitutionsare at positions selected from 16, 26, 29, 33, 91, 114, 122, 128, 130,145, 157, 158, 183, 256, 270, 272, 302, 322, 371, 383, and 394, 412. Insome embodiments, the one or more substitutions are at positionsselected from 16, 33, 157, 256, 302, 371, and 383.

In some embodiments, the one or more substitutions are selected from 2Kor L; 6I; 16D; 18S; 19E; 24V; 26A; 27L; 29G, R, S, A, or E; 31L; 33E;37T or R; 38I; 39I; 41F; 45S; 48D; 49A; 51R; 52R; 58T or A; 61Y; 66L;72T; 73V; 74A; 81E; 82A; 91H; 92A, E, G, or Q; 94G; 95L or V; 98I; 103Dor L; 111A; 112Q; 114H or R; 122P, or R; 123E, C, G, or R; 124N or R;126C, D, E, K, L, M, N, Q, W, R, or S; 127E, I, R, S, V, or W; 128W;129C or W; 130D; 131H; R, or Y; 132V; 134A, F, or R; 137A; 140K or V;141F; 145G; 149K, R, or T; 157Y; 158R; 165V; 166L; 167W or Y; 170S;171I; 172K or R; 178I; 180E, P, or Q; 183E, N, or V; 184D or K; 189D;194F, E, or R; 203S; 204D; 205V; 206E; 210G or P; 214G, L, or R; 216L;220G; 221T or V; 223L; 224L; 225G, D, or T; 226T; 227C or V; 228V; 246Ror T; 250D; 252I; 253R; 254G or S; 255M or R; 256S; 257E, G, R, S, or V;258Q or S; 259C, D, Q, R, or W; 268T; 269F; 270D; 272G, K, P, R, or S;275I or R; 276Q, A, or K; 279L, R, or V; 280C or L; 282R or T; 284C, L,M, R, S, or Y; 285V; 286T; 294H, A, or L; 295N; 298H, K, M, Q, D, or N;301K; 302Q, G, H, R, V, or W; 304T; 305Q, R, or W; 306F; 308L; 309A orS; 310R; 313D; 315S; 317G; 319L or S; 322R or A; 324T; 331P; 338S; 345A;346G; 349R; 359G; 360G; 366L; 371F; 374S; 380R; 383E, or L; 384M; 385L;387G or V; 388E; 392C; 393R, S, or W; 394E, G, N, R, or S; 396H, E, orQ; 397M or T; 409A, R, S, or W; 411E; 412A, L, D, or S; 413Y; 415C or G;417W; 420V; 423V; 428E or T; 429S; 431T; 437L; 441R or W; 442E, R, or W;444G, R, or V; 446G; 448P, Q or T; 451F; 455L, R, T, or W; 458F; 459W;463M; 464L; 466C or E; 467P or W; 468I, T, or V; 469I; 470S, G, L, S, orT; 473D or M; 474G; 480P, T, or L; 481V; 482L; 483A, Q, or T; 491L; 492Gor R; 493D; 494C; and 495E, G, L, or Y. In some embodiments, the aminoacid positions of the polypeptide sequence are numbered with referenceto SEQ ID NO: 2 or 18. In some further embodiments, the amino acidpositions of the polypeptide sequence are numbered with reference to SEQID NO: 40, 156, 174, 276, 308, 422, 456, 546, 632, 664, 780, 988, 1104,1122, 1224, 1256, 1370, 1404, 1494, 1580, 1612, or 1728, a furtherdescribed herein.

In some embodiments, the recombinant amylase and/or biologically activerecombinant amylase fragment comprises a polypeptide sequence having atleast 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 18, 40,156, 174, 276, 308, 422, 456, 546, 632, 664, 780, 988, 1104, 1122, 1224,1256, 1370, 1404, 1494, 1580, 1612, or 1728. In some embodiments, therecombinant amylase and/or biologically active recombinant amylasefragment comprises a polypeptide sequence having at least 70%, 75%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or more sequence identity to SEQ ID NO: 18, 40, 156, 174, 276, 308,422, 456, 546, 632, 664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404,1494, 1580, 1612, or 1728, with the proviso that recombinant amylasedoes not have the polypeptide sequence of SEQ ID NO:2.

In some embodiments, the recombinant amylase and/or biologically activerecombinant amylase fragment comprises a polypeptide sequence having atleast 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 18, 40,156, 174, 276, 308, 422, 456, 546, 632, 664, 780, 988, 1104, 1122, 1224,1256, 1370, 1404, 1494, 1580, 1612, or 1728, and further comprises oneor more substitutions. In some embodiments, the recombinant amylaseand/or biologically active recombinant amylase fragment comprises apolypeptide sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequenceidentity to SEQ ID NO: 18, 40, 156, 174, 276, 308, 422, 456, 546, 632,664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494, 1580, 1612, or1728, and further comprises one or more substitutions at positionsselected from 2, 6, 16, 18, 19, 24, 26, 27, 29, 31, 33, 37, 38, 39, 41,45, 48, 49, 51, 52, 58, 61, 66, 72, 73, 74, 81, 82, 91, 92, 94, 95, 98,103, 111, 112, 114, 122, 123, 124, 126, 127, 128, 129, 130, 131, 132,134, 137, 140, 141, 145, 149, 157, 158, 165, 166, 167, 170, 171, 172,178, 180, 183, 184, 189, 194, 203, 204, 205, 206, 210, 214, 216, 220,221, 223, 224, 225, 226, 227, 228, 246, 250, 252, 253, 254, 255, 256,257, 258, 259, 268, 269, 270, 272, 275, 276, 279, 280, 282, 284, 285,286, 294, 295, 298, 301, 302, 304, 305, 306, 308, 309, 310, 313, 315,317, 319, 322, 324, 331, 338S, 345, 346, 349, 359, 360, 366, 371, 374,380, 383, 384, 385, 387, 388, 392, 393, 394, 396, 397, 409, 411, 412,413, 415, 417, 420, 423, 428, 429, 431, 437L 441, 442, 444, 446, 448,451, 455, 458, 459, 463, 464, 466, 467, 468, 469, 470, 473, 474, 480,481, 482, 483, 491, 492, 493, 494, and 495, wherein the amino acidpositions of the polypeptide sequence are numbered with reference to SEQID NO: 18, 40, 156, 174, 276, 308, 422, 456, 546, 632, 664, 780, 988,1104, 1122, 1224, 1256, 1370, 1404, 1494, 1580, 1612, or 1728. In someembodiments, the amino acid substitutions described above for thepositions can be used.

In some additional embodiments, the recombinant amylase comprises apolypeptide sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequenceidentity to SEQ ID NO: 2 or 18, or a functional fragment thereof, andwherein the recombinant amylase comprises one or more substitutions atpositions selected from 16, 26, 29, 33, 91, 114, 157, 158, 180, 184,214, 223, 256, 272, 298, 302, 371, 380, 383, 394 412, 428, and 437,wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 2 or 18.

In some additional embodiments, the recombinant amylase comprises apolypeptide sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequenceidentity to SEQ ID NO: 2 or 18, or a functional fragment thereof, andwherein the recombinant amylase comprises one or more substitutionsselected from 16D, 26A, 29G, 29R, 29S, 29A, 29E, 33E, 91H, 114H, 157Y,158R, 180E, 180Q, 184D, 184K, 214G, 223L, 256T, 272G, 272K, 272P, 272R,272S, 298H, 302Q, 302G, 302H, 302R, 302V, 302W, 371F, 380R, 383E, 383I,394N, 394S, 394G, 412A, 412L, 412D, 412S, 428T, and 437L, wherein theamino acid positions of the polypeptide sequence are numbered withreference to SEQ ID NO: 2 or 18.

In some additional embodiments, the recombinant amylase comprises apolypeptide sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequenceidentity to SEQ ID NO: 2 or 18, or a functional fragment thereof, andwherein the recombinant amylase comprises at least one substitution orsubstitution set at one or more positions selected from 2/73/256/493,16/33/73/130/256/282/371/374/493, 16/33/130/256/302/371/383/470,16/33/157/256/302/371/383, 16/73/74/256/282/366/371/383/493,16/73/256/282/302/331/371/383, 16/130/256/276/374/470, 16/256/302/374,19/72/345/473, 26/27/396/412/428/473, 26/27/412, 26/27/466/473, 29,73/130/256/282/302/493, 74/371/374/383/493, 130/256/493, 149, 158, 254,256, 256/383, 257, 259, 272, 279, 284, 317/383, 322, 345/388/396/428,388/396/412/428, 396/412, 396/412/428/466, 409, 412, and 495, whereinthe amino acid positions of the polypeptide sequence are numbered withreference to SEQ ID NO: 2 or 18. In some further embodiments, therecombinant amylase comprises a polypeptide sequence having at least70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 2 or 18, ora functional fragment thereof, and wherein the recombinant amylasecomprises at least one substitution or substitution set selected from2K/73V/256S/493D, 16D/33E/73V/130D/256S/282D/371F/374S/493D,16D/33E/130D/256S/302Q/371F/383E/470T, 16D/33E/157Y/256S/302Q/371F/383E,16D/73V/74A/256S/282D/366L/371F/383E/493D,16D/73V/256S/282D/302Q/331P/371F/383E, 16D/130D/256S/276Q/374S/470T,16D/256S/302Q/374S, 19E/72T/345A/473D, 26A/27L/396H/412D/428E/473D,26A/27L/412D, 26A/27L/466E/473D, 29G, 29R, 29S,73V/130D/256S/282D/302Q/493D, 74A/371F/374S/383E/493D, 130D/256S/493D,149R, 149T, 158R, 254G, 254S, 256S, 256S/383E, 257E, 257G, 257R, 257S,257V, 259Q, 272G, 272K, 272P, 272R, 272S, 279R, 284C, 284R, 284S, 284Y,317G/383L, 322A, 345A/388E/396H/428E, 388E/396H/412D/428E, 396H/412D,396H/412D/428E/466E, 409S, 412S, 495E, 495L, and 495Y, wherein the aminoacid positions of the polypeptide sequence are numbered with referenceto SEQ ID NO: 2 or 18. In some further embodiments, the recombinantamylase comprises a polypeptide sequence having at least 70%, 75%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or more sequence identity to SEQ ID NO: 2 or 18, or a functionalfragment thereof, and wherein the recombinant amylase comprises at leastone substitution or substitution set selected from R2K/I73V/A256S/E493D,E16D/D33E/I73V/E130D/A256S/E282D/Y371F/H374S/E493D,E16D/D33E/E130D/A256S/M302Q/Y371F/Q383E/V470T,E16D/D33E/F157Y/A256S/M302Q/Y371F/Q383E,E16D/I73V/P74A/A256S/E282D/F366L/Y371F/Q383E/E493D,E16D/I73V/A256S/E282D/M302Q/H331P/Y371F/Q383E,E16D/E130D/A256S/T276Q/H374S/V470T, E16D/A256S/M302Q/H374S,K19E/S72T/P345A/E473D, E26A/R27L/D396H/L412D/D428E/E473D,E26A/R27L/L412D, E26A/R27L/Q466E/E473D, D29G, D29R, D29S,I73V/E130D/A256S/E282D/M302Q/E493D, P74A/Y371F/H374S/Q383E/E493D,E130D/A256S/E493D, Q149R, Q149T, K158R, E254G, E254S, A256S,A256S/Q383E, P257E, P257G, P257R, P257S, P257V, P259Q, D272G, D272K,D272P, D272R, D272S, D279R, K284C, K284R, K284S, K284Y, V317G/Q383L,F322A, P345A/D388E/D396H/D428E, D388E/D396H/L412D/D428E, D396H/L412D,D396H/L412D/D428E/Q466E, V409S, L412S, 1495E, I495L, and I495Y, whereinthe amino acid positions of the polypeptide sequence are numbered withreference to SEQ ID NO: 2 or 18.

In some additional embodiments, the recombinant amylase comprises apolypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO: 40 or 988, or a functional fragment thereof, and whereinthe recombinant amylase comprises at least one substitution orsubstitution set at one or more positions selected from26/29/158/272/412, 26/29/272/279/412/493, 26/29/272/396/412,26/29/279/412/428, 26/158/272/279/388/412/428, 26/158/272/412,26/272/279/412/428/493, 29/272/279/396/412, 130/149/254/284/322/409/495,130/149/257/284/322/409/466/473, 130/254/284/322/374/409/466,130/257/284/374/409/466, 130/284/322/374/409/473,149/257/284/322/409/466, 158/272/279/394/396/412/428/493,158/272/388/412/428/493, 158/272/412/428, 158/279/388/396/412/428,254/257/284/322/409/473, 254/284/409/466/495, 272/279/412/428/493,272/412/428, 272/412/493, and 284/322/409/473, wherein the amino acidpositions of the polypeptide sequence are numbered with reference to SEQID NO: 40 or 988. In some further embodiments, the recombinant amylasecomprises a polypeptide sequence having at least 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NO: 40 or 988, or a functional fragmentthereof, and wherein the recombinant amylase comprises at least onesubstitution or substitution set selected from 26A/29R/158R/272G/412D,26A/29R/272G/279R/412D/493D, 26A/29R/272G/396H/412D,26A/29R/279R/412D/428E, 26A/158R/272G/279R/388E/412D/428E,26A/158R/272G/412D, 26A/272G/279R/412D/428E/493D,29R/272G/279R/396H/412D, 130D/149R/254S/284S/322A/409S/495E,130D/149R/257G/284S/322A/409S/466E/473D,130D/254S/284S/322A/374S/409S/466E, 130D/257G/284S/374S/409S/466E,130D/284S/322A/374S/409S/473D, 149R/257G/284S/322A/409S/466E,158R/272G/279R/394R/396H/412D/428E/493D, 158R/272G/388E/412D/428E/493D,158R/272G/412D/428E, 158R/279R/388E/396H/412D/428E,254S/257G/284S/322A/409S/473D, 254S/284S/409S/466E/495E,272G/279R/412D/428E/493D, 272G/412D/428E, 272G/412D/493D, and284S/322A/409S/473D, wherein the amino acid positions of the polypeptidesequence are numbered with reference to SEQ ID NO: 40 or 988. In somefurther embodiments, the recombinant amylase comprises a polypeptidesequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ IDNO: 40 or 988, or a functional fragment thereof, and wherein therecombinant amylase comprises at least one substitution or substitutionset selected from E26A/D29R/K158R/D272G/L412D,E26A/D29R/D272G/D279R/L412D/E493D, E26A/D29R/D272G/D396H/L412D,E26A/D29R/D279R/L412D/D428E, E26A/K158R/D272G/D279R/D388E/L412D/D428E,E26A/K158R/D272G/L412D, E26A/D272G/D279R/L412D/D428E/E493D,D29R/D272G/D279R/D396H/L412D, E130D/Q149R/E254S/K284S/F322A/V409S/I495E,E130D/Q149R/P257G/K284S/F322A/V409S/Q466E/E473D,E130D/E254S/K284S/F322A/H374S/V409S/Q466E,E130D/P257G/K284S/H374S/V409S/Q466E,E130D/K284S/F322A/H374S/V409S/E473D,Q149R/P257G/K284S/F322A/V409S/Q466E,K158R/D272G/D279R/Q394R/D396H/L412D/D428E/E493D,K158R/D272G/D388E/L412D/D428E/E493D, K158R/D272G/L412D/D428E,K158R/D279R/D388E/D396H/L412D/D428E,E254S/P257G/K284S/F322A/V409S/E473D, E254S/K284S/V409S/Q466E/I495E,D272G/D279R/L412D/D428E/E493D, D272G/L412D/D428E, D272G/L412D/E493D, andK284S/F322A/V409S/E473D, wherein the amino acid positions of thepolypeptide sequence are numbered with reference to SEQ ID NO: 40 or988.

In some additional embodiments, the recombinant amylase comprises apolypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO: 156 or 1104, or a functional fragment thereof, and whereinthe recombinant amylase comprises at least one substitution orsubstitution set at one or more positions selected from 37, 51, 91, 111,112, 114, 122, 124, 128, 140, 167, 171, 183, 194, 276, 280, 298, 397,and 464, wherein the amino acid positions of the polypeptide sequenceare numbered with reference to SEQ ID NO: 156 or 1104. In some furtherembodiments, the recombinant amylase comprises a polypeptide sequencehaving at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 156 or1104, or a functional fragment thereof, and wherein the recombinantamylase comprises at least one substitution or substitution set selectedfrom 37R, 51R, 91H, 111A, 112Q, 114H, 122P, 122R, 124R, 128W, 140K,167W, 171I, 183E, 183N, 183V, 194E, 194R, 276A, 276K, 280L, 298K, 298M,397T, and 464L, wherein the amino acid positions of the polypeptidesequence are numbered with reference to SEQ ID NO: 156 or 1104. In somefurther embodiments, the recombinant amylase comprises a polypeptidesequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ IDNO: 156 or 1104, or a functional fragment thereof, and wherein therecombinant amylase comprises at least one substitution or substitutionset selected from N37R, G51R, K91H, E111A, K112Q, A114H, A122P, A122R,D124R, I128W, R140K, E167W, E171I, G183E, G183N, G183V, L194E, L194R,T276A, T276K, Q280L, E298K, E298M, E397T, and N464L, wherein the aminoacid positions of the polypeptide sequence are numbered with referenceto SEQ ID NO: 156 or 1104.

In some additional embodiments, the recombinant amylase comprises apolypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO: 174 or 1122, or a functional fragment thereof, and whereinthe recombinant amylase comprises at least one substitution orsubstitution set at one or more positions selected from 24, 91, 91/122,91/122/128/130/167/171/409, 91/122/128/130/183/322, 91/122/128/130/194,91/122/128/130/322, 91/122/128/171/183/194/322, 91/122/128/183/409,91/122/130/167/183/194/409, 91/122/130/171/183, 91/122/130/183/194,91/122/130/194, 91/122/130/194/322, 91/122/167/171/194,91/122/167/171/194/322, 91/122/171/183/194/409, 91/130/171/183/322/409,91/167/171, 91/167/183/194, 91/167/322, 91/183, 91/183/322/409, 91/194,91/322, 91/409, 122/128, 122/128/194/409, 122/130/171/322/409, 122/409,137/145/227/394/480/481, 137/145/295, 145/227/295/481, 145/309/481, 183,227, 295/394, 322, 322/409, 394, and 480/481, wherein the amino acidpositions of the polypeptide sequence are numbered with reference to SEQID NO: 174 or 1122. In some further embodiments, the recombinant amylasecomprises a polypeptide sequence having at least 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NO: 174 or 1122, or a functional fragmentthereof, and wherein the recombinant amylase comprises at least onesubstitution or substitution set selected from 24V, 91H, 91H/122P,91H/122P/130D/183N/194R, 91H/122P/130D/194R/322A, 91H/122R,91H/122R/128W/130D/167W/171I/409S, 91H/122R/128W/130D/183N/322A,91H/122R/128W/130D/194R, 91H/122R/128W/130D/322A,91H/122R/128W/171I/183N/194R/322A, 91H/122R/128W/183N/409S,91H/122R/130D/167W/183N/194R/409S, 91H/122R/130D/171I/183N,91H/122R/130D/194R, 91H/122R/167W/171I/194R,91H/122R/167W/171I/194R/322A, 91H/122R/171I/183N/194R/409S,91H/130D/171I/183N/322A/409S, 91H/167W/171I, 91H/167W/183N/194R,91H/167W/322A, 91H/183N, 91H/183N/322A/409S, 91H/194R, 91H/322A,91H/409S, 122P/409S, 122R/128W, 122R/128W/194R/409S,122R/130D/171I/322A/409S, 137A/145G/227L/394E/480P/481V, 137A/145G/295N,145G/227L/295N/481V, 145G/309S/481V, 183N, 227L, 295N/394E, 322A,322A/409S, 394E, and 480T/481V, wherein the amino acid positions of thepolypeptide sequence are numbered with reference to SEQ ID NO: 174 or1122. In some further embodiments, the recombinant amylase comprises apolypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO: 174 or 1122, or a functional fragment thereof, and whereinthe recombinant amylase comprises at least one substitution orsubstitution set selected from L24V, K91H, K91H/A122P,K91H/A122P/E130D/G183N/L194R, K91H/A122P/E130D/L194R/F322A, K91H/A122R,K91H/A122R/I128W/E130D/E167W/E171I/V409S,K91H/A122R/I128W/E130D/G183N/F322A, K91H/A122R/I128W/E130D/L194R,K91H/A122R/I128W/E130D/F322A, K91H/A122R/I128W/E171I/G183N/L194R/F322A,K91H/A122R/I128W/G183N/V409S, K91H/A122R/E130D/E167W/G183N/L194R/V409S,K91H/A122R/E130D/E171I/G183N, K91H/A122R/E130D/L194R,K91H/A122R/E167W/E171I/L194R, K91H/A122R/E167W/E171I/L194R/F322A,K91H/A122R/E171I/G183N/L194R/V409S, K91H/E130D/E171I/G183N/F322A/V409S,K91H/E167W/E171I, K91H/E167W/G183N/L194R, K91H/E167W/F322A, K91H/G183N,K91H/G183N/F322A/V409S, K91H/L194R, K91H/F322A, K91H/V409S, A122P/V409S,A122R/1128W, A122R/1128W/L194R/V409S, A122R/E130D/E171I/F322A/V409S,G137A/A145G/T227L/Q394E/F480P/C481V, G137A/A145G/K295N,A145G/T227L/K295N/C481V, A145G/T309S/C481V, G183N, T227L, K295N/Q394E,F322A, F322A/V409S, Q394E, and F480T/C481V, wherein the amino acidpositions of the polypeptide sequence are numbered with reference to SEQID NO: 174 or 1122.

In some additional embodiments, the recombinant amylase comprises apolypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO: 276 or 1224, or a functional fragment thereof, and whereinthe recombinant amylase comprises at least one substitution orsubstitution set at one or more positions selected from16/26/29/128/145/256/309/383, 16/26/33/114/145/309/383/394,16/26/33/128/130/145/302/309/322/371, 16/26/33/145/158/383/394,16/26/145/302, 16/29/33/145/256/302, 18,26/29/33/130/145/157/158/183/394, 26/29/33/145/157/183/256/309/383,26/29/122/130/145/183/256/272/309/394, 26/29/145/157/256/302/322/481,26/29/145/256/272/302/309/322/481, 26/33/91/145/272/302/481,26/33/145/309/322/383, 26/114/145/322, 26/145/183/256/309/383,29/122/145/158/383, 29/130/145/256/272/371/394, 29/145/183/302/309/412,29/309/394, 33/122/145/272/309/412, 91/130/145/158/183/383, 91/309/383,130/145/256/272/371, 130/145/302/309, 130/272/481, 145/302/309, 268,309, 349, 360, 380, 394, 463, and 483, wherein the amino acid positionsof the polypeptide sequence are numbered with reference to SEQ ID NO:276 or 1224. In some further embodiments, the recombinant amylasecomprises a polypeptide sequence having at least 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NO: 276 or 1224, or a functional fragmentthereof, and wherein the recombinant amylase comprises at least onesubstitution or substitution set selected from16E/26E/29D/128I/145G/256A/309S/383Q,16E/26E/33D/114A/145G/309S/383Q/394E,16E/26E/33D/128I130E/145G/302M/309S/322F/371Y,16E/26E/33D/145G/158K/383Q/394E, 16E/26E/145G/302M,16E/29D/33D/145G/256A/302M, 18S,26E/29D/33D/130E/145G/157F/158K/183G/394E,26E/29D/33D/145G/157F/183G/256A/309S/383Q,26E/29D/122A/130E/145G/183G/256A/272D/309S/394E,26E/29D/145G/157F/256A/302M/322F/481V,26E/29D/145G/256A/272D/302M/309S/322F/481V,26E/33D/91K/145G/272D/302M/481V, 26E/33D/145G/309S/322F/383Q,26E/114A/145G/322F, 26E/145G/183G/256A/309S/383Q,29D/122A/145G/158K/383Q, 29D/130E/145G/256A/272D/371Y/394E,29D/145G/183G/302M/309S/412L, 29D/309S/394E,33D/122A/145G/272D/309S/412L, 91K/130E/145G/158K/183G/383Q,91K/309S/383Q, 130E/145G/256A/272D/371Y, 130E/145G/302M/309S,130E/272D/481V, 145G/302M/309S, 268T, 309A, 349R, 360G, 380R, 394G,394N, 394S, 463M, and 483T, wherein the amino acid positions of thepolypeptide sequence are numbered with reference to SEQ ID NO: 276 or1224. In some further embodiments, the recombinant amylase comprises apolypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO: 276 or 1224, or a functional fragment thereof, and whereinthe recombinant amylase comprises at least one substitution orsubstitution set selected fromD16E/A26E/R29D/W128I/A145G/S256A/T309S/E383Q,D16E/A26E/E33D/H114A/A145G/T309S/E383Q/Q394E,D16E/A26E/E33D/W128I/D130E/A145G/Q302M/T309S/A322F/F371Y,D16E/A26E/E33D/A145G/R158K/E383Q/Q394E, D16E/A26E/A145G/Q302M,D16E/R29D/E33D/A145G/S256A/Q302M, G18S,A26E/R29D/E33D/D130E/A145G/Y157F/R158K/N183G/Q394E,A26E/R29D/E33D/A145G/Y157F/N183G/S256A/T309S/E383Q,A26E/R29D/R122A/D130E/A145G/N183G/S256A/G272D/T309S/Q394E,A26E/R29D/A145G/Y157F/S256A/Q302M/A322F/C481V,A26E/R29D/A145G/S256A/G272D/Q302M/T309S/A322F/C481V,A26E/E33D/H91K/A145G/G272D/Q302M/C481V,A26E/E33D/A145G/T309S/A322F/E383Q, A26E/H114A/A145G/A322F,A26E/A145G/N183G/S256A/T309S/E383Q, R29D/R122A/A145G/R158K/E383Q,R29D/D130E/A145G/S256A/G272D/F371Y/Q394E,R29D/A145G/N183G/Q302M/T309S/D412L, R29D/T309S/Q394E,E33D/R122A/A145G/G272D/T309S/D412L, H91K/D130E/A145G/R158K/N183G/E383Q,H91K/T309S/E383Q, D130E/A145G/S256A/G272D/F371Y,D130E/A145G/Q302M/T309S, D130E/G272D/C481V, A145G/Q302M/T309S, S268T,T309A, P349R, N360G, G380R, Q394G, Q394N, Q394S, G463M, and G483T,wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 276 or 1224.

In some additional embodiments, the recombinant amylase comprises apolypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO: 308 or 1256, or a functional fragment thereof, and whereinthe recombinant amylase comprises at least one substitution orsubstitution set at one or more positions selected from 29,37/124/250/270/349/380/394/442/491, 37/145, 37/145/250/301/349/394,37/145/258/442/491, 38/349/491, 81/259, 81/313/495, 92/257/258/338/468,92/258, 94/127/132/145/250/349, 94/270/301/349/394/442, 94/349/491,145/270, 178, 225, 226, 246, 250/349/442, 259/313/338, 270/349, 286/338,294, 313, 349, 349/380/442, 413, 413/468, 448, and 473, wherein theamino acid positions of the polypeptide sequence are numbered withreference to SEQ ID NO: 308 or 1256. In some further embodiments, therecombinant amylase comprises a polypeptide sequence having at least80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or more sequence identity to SEQ ID NO: 308 or 1256, or afunctional fragment thereof, and wherein the recombinant amylasecomprises at least one substitution or substitution set selected from29A, 37T/124N/250D/270D/349R/380R/394E/442E/491L, 37T/145G,37T/145G/250D/301K/349R/394E, 37T/145G/258S/442E/491L, 38I/349R/491L,81E/259D, 81E/313D/495L, 92E/257E/258Q/338S/468T, 92E/258Q,94G/127E/132V/145G/250D/349R, 94G/270D/301K/349R/394E/442E,94G/349R/491L, 145G/270D, 178I, 225D, 226T, 246R, 250D/349R/442E,259D/313D/338S, 270D/349R, 286T/338S, 294L, 313D, 349R, 349R/380R/442E,413Y, 413Y/468I, 448T, and 473D, wherein the amino acid positions of thepolypeptide sequence are numbered with reference to SEQ ID NO: 308 or1256. In some further embodiments, the recombinant amylase comprises apolypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO: 308 or 1256, or a functional fragment thereof, and whereinthe recombinant amylase comprises at least one substitution orsubstitution set selected from R29A,N37T/D124N/E250D/E270D/P349R/G380R/G394E/D442E/I491L, N37T/A145G,N37T/A145G/E250D/R301K/P349R/G394E, N37T/A145G/K258S/D442E/I491L,M38I/P349R/I491L, A81E/P259D, A81E/T313D/I495L,R92E/P257E/K258Q/A338S/R468T, R92E/K258Q,D94G/Q127E/I132V/A145G/E250D/P349R, D94G/E270D/R301K/P349R/G394E/D442E,D94G/P349R/I491L, A145G/E270D, V178I, E225D, Q226T, K246R,E250D/P349R/D442E, P259D/T313D/A338S, E270D/P349R, M286T/A338S, M294L,T313D, P349R, P349R/G380R/D442E, F413Y, F413Y/R468I, H448T, and E473D,wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 308 or 1256.

In some additional embodiments, the recombinant amylase comprises apolypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO: 422 or 1370, or a functional fragment thereof, and whereinthe recombinant amylase comprises at least one substitution orsubstitution set at one or more positions selected from 38,38/178/226/286/338, 38/178/286/448, 38/178/349/448, 38/226/246/258/286,38/226/258/286/338/380/448, 38/226/258/349/413, 38/226/286,38/226/286/338/380, 38/226/338/349/380, 38/226/413/448, 38/246/286,38/246/286/380, 38/258, 38/286, 38/286/413/448, 38/286/448, 38/448, 178,178/226/258/286/310/338, 178/226/286/338, 178/246/338, 178/258/286/413,178/286, 178/286/338/349, 178/286/338/448, 178/380, 178/413, 178/448,184/246/258/380, 226, 226/246/286/349/448, 226/246/413,226/258/286/349/380, 226/258/286/413/448, 226/286, 226/286/380/448,226/349/380, 246, 246/286, 246/286/380/413, 246/338, 246/413,258/286/413, 286, 286/413/448, 338/448, 349, and 413, wherein the aminoacid positions of the polypeptide sequence are numbered with referenceto SEQ ID NO: 422 or 1370. In some further embodiments, the recombinantamylase comprises a polypeptide sequence having at least 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NO: 422 or 1370, or a functional fragmentthereof, and wherein the recombinant amylase comprises at least onesubstitution or substitution set selected from 38I,38I/178I/226T/286T/338S, 38I/178I/286T/448T, 38I/178I/349R/448T,38I/226T/246R/258S/286T, 38I/226T/258S/286T/338S/380R/448T,38I/226T/258S/349R/413Y, 38I/226T/286T, 38I/226T/286T/338S/380R,38I/226T/338S/349R/380R, 38I/226T/413Y/448T, 38I/246R/286T,38I/246R/286T/380R, 38I/258S, 38I/286T, 38I/286T/413Y/448T,38I/286T/448T, 38I/448T, 178I, 178I/226T/258S/286T/310R/338S,178I/226T/286T/338S, 178I/246R/338S, 178I/258S/286T/413Y, 178I/286T,178I/286T/338S/349R, 178I/286T/338S/448T, 178I/380R, 178I/413Y,178I/448T, 184K/246R/258S/380R, 226T, 226T/246R/286T/349R/448T,226T/246R/413Y, 226T/258S/286T/349R/380R, 226T/258S/286T/413Y/448T,226T/286T, 226T/286T/380R/448T, 226T/349R/380R, 246R, 246R/286T,246R/286T/380R/413Y, 246R/338S, 246R/413Y, 246T, 258S/286T/413Y, 286T,286T/413Y/448T, 338S/448T, 349R, and 413Y, wherein the amino acidpositions of the polypeptide sequence are numbered with reference to SEQID NO: 422 or 1370. In some further embodiments, the recombinant amylasecomprises a polypeptide sequence having at least 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NO: 422 or 1370, or a functional fragmentthereof, and wherein the recombinant amylase comprises at least onesubstitution or substitution set selected from M38I,M38I/V178I/Q226T/M286T/A338S, M38I/V178I/M286T/H448T,M38I/V178I/P349R/H448T, M38I/Q226T/K246R/K258S/M286T,M38I/Q226T/K258S/M286T/A338S/G380R/H448T, M38I/Q226T/K258S/P349R/F413Y,M38I/Q226T/M286T, M38I/Q226T/M286T/A338S/G380R,M38I/Q226T/A338S/P349R/G380R, M38I/Q226T/F413Y/H448T, M38I/K246R/M286T,M38I/K246R/M286T/G380R, M38I/K258S, M38I/M286T, M38I/M286T/F413Y/H448T,M38I/M286T/H448T, M38/1H448T, V178I,V178I/Q226T/K258S/M286T/Q310R/A338S, V178I/Q226T/M286T/A338S,V178I/K246R/A338S, V178I/K258S/M286T/F413Y, V178I/M286T,V178I/M286T/A338S/P349R, V178I/M286T/A338S/H448T, V178I/G380R,V178I/F413Y, V178/1H448T, E184K/K246R/K258S/G380R, Q226T,Q226T/K246R/M286T/P349R/H448T, Q226T/K246R/F413Y,Q226T/K258S/M286T/P349R/G380R, Q226T/K258S/M286T/F413Y/H448T,Q226T/M286T, Q226T/M286T/G380R/H448T, Q226T/P349R/G380R, K246R,K246R/M286T, K246R/M286T/G380R/F413Y, K246R/A338S, K246R/F413Y, K246T,K258S/M286T/F413Y, M286T, M286T/F413Y/H448T, A338S/H448T, P349R, andF413Y, wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 422 or 1370.

In some additional embodiments, the recombinant amylase comprises apolypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO: 456 or 1404, or a functional fragment thereof, and whereinthe recombinant amylase comprises at least one substitution orsubstitution set at one or more positions selected from 29/31/92/220,29/469, 49/58/103/141/197/269/294/298, 49/58/103/194/204/298,49/58/141/298, 49/103, 103, 123, 126, 127, 131, 134, 180, 210, 214,220/469, 228, 259, 279, 282, 302, 319, 393, 415, 417, 428, 429, 466, and469, wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 456 or 1404. In some furtherembodiments, the recombinant amylase comprises a polypeptide sequencehaving at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 456 or1404, or a functional fragment thereof, and wherein the recombinantamylase comprises at least one substitution or substitution set selectedfrom 29E/31L/92A/220G, 29E/469I, 49A/58T/103L/141F/197Y/269F/294H/298Q,49A/58T/103L/194F/204D/298H, 49A/58T/141F/298H, 49A/103L, 103L, 123R,126R, 127W, 131H, 131R, 131Y, 134R, 180E, 210P, 214G, 220G/469I, 228V,259C, 279L, 282T, 302H, 302R, 302W, 319S, 393R, 393S, 415C, 417W, 428T,429S, 466C, and 469I, wherein the amino acid positions of thepolypeptide sequence are numbered with reference to SEQ ID NO: 456 or1404. In some further embodiments, the recombinant amylase comprises apolypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO: 456 or 1404, or a functional fragment thereof, and whereinthe recombinant amylase comprises at least one substitution orsubstitution set selected from R29E/F31L/R92A/A220G, R29E/V469I,S49A/S58T/V103L/Y141F/F197Y/H269F/M294H/E298Q,S49A/S58T/V103L/L194F/N204D/E298H, S49A/S58T/Y141F/E298H, S49A/V103L,V103L, D123R, T126R, Q127W, E131H, E131R, E131Y, E134R, D180E, H210P,E214G, A220G/V469I, Q228V, P259C, D279L, E282T, Q302H, Q302R, Q302W,A319S, E393R, E393S, D415C, P417W, D428T, E429S, Q466C, and V469I,wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 456 or 1404.

In some additional embodiments, the recombinant amylase comprises apolypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO: 456 or 1404, or a functional fragment thereof, and whereinthe recombinant amylase comprises at least one substitution orsubstitution set at one or more positions selected from 92, 123, 126,127, 129, 131, 134, 140, 172, 180, 210, 214, 221, 228, 253, 254, 259,279, 282, 302, 304, 305, 319, 322, 346, 359, 384, 385, 387, 393, 409,412, 415, 417, 428, 429, 441, 442, 444, 446, 451, 455, 459, 466, 467,470, 473, 474, 492, and 494, wherein the amino acid positions of thepolypeptide sequence are numbered with reference to SEQ ID NO: 456 or1404. In some further embodiments, the recombinant amylase comprises apolypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO: 456 or 1404, or a functional fragment thereof, and whereinthe recombinant amylase comprises at least one substitution orsubstitution set selected from 92G, 92Q, 123C, 123G, 123R, 126Q, 126R,1271, 127R, 127S, 127V, 127W, 129C, 129W, 131H, 131R, 131Y, 134A, 134F,134R, 140V, 172K, 172R, 180E, 180P, 180Q, 210G, 210P, 214G, 214L, 214R,221T, 221V, 228V, 253R, 254G, 259C, 259R, 259W, 279L, 279V, 282R, 282T,302G, 302H, 302R, 302V, 302W, 304T, 305Q, 305R, 305W, 319L, 319S, 322R,346G, 359G, 384M, 385L, 387G, 387V, 393R, 393S, 393W, 409A, 409R, 409W,412A, 415C, 415G, 417W, 428T, 429S, 441R, 441W, 442R, 442W, 444G, 444R,444V, 446G, 451F, 455L, 455R, 455T, 455W, 459W, 466C, 467P, 467W, 470G,470L, 470S, 473M, 474G, 492G, 492R, and 494C, wherein the amino acidpositions of the polypeptide sequence are numbered with reference to SEQID NO: 456 or 1404. In some further embodiments, the recombinant amylasecomprises a polypeptide sequence having at least 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NO: 456 or 1404, or a functional fragmentthereof, and wherein the recombinant amylase comprises at least onesubstitution or substitution set selected from R92G, R92Q, D123C, D123G,D123R, T126Q, T126R, Q1271, Q127R, Q127S, Q127V, Q127W, D129C, D129W,E131H, E131R, E131Y, E134A, E134F, E134R, R140V, D172K, D172R, D180E,D180P, D180Q, H210G, H210P, E214G, E214L, E214R, R221T, R221V, Q228V,Q253R, E254G, P259C, P259R, P259W, D279L, D279V, E282R, E282T, Q302G,Q302H, Q302R, Q302V, Q302W, R304T, D305Q, D305R, D305W, A319L, A319S,A322R, W346G, E359G, T384M, Y385L, 1387G, 1387V, E393R, E393S, E393W,V409A, V409R, V409W, D412A, D415C, D415G, P417W, D428T, E429S, D441R,D441W, D442R, D442W, E444G, E444R, E444V, T446G, E451F, N455L, N455R,N455T, N455W, R459W, Q466C, E467P, E467W, V470G, V470L, V470S, E473M,N474G, E492G, E492R, and V494C, wherein the amino acid positions of thepolypeptide sequence are numbered with reference to SEQ ID NO: 456 or1404.

In some additional embodiments, the recombinant amylase comprises apolypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO: 546 or 1494, or a functional fragment thereof, and whereinthe recombinant amylase comprises at least one substitution orsubstitution set at one or more positions selected from 123,123/126/180/214/298/302/393, 123/126/180/214/393/428,123/126/210/298/302/393, 123/126/210/428, 123/126/428, 123/210/302,126/180/210/214/298/302, 126/180/210/214/428, 126/180/298/302/393,126/210/302/428, 126/214/393, 126/298/302/393, 126/298/302/393/428, 129,129/228/253, 131/228/279, 131/282, 180/214, 210/214/302/393/428,228/282/444, 253/279/470, 253/279/492, 279, 279/470, and 441, whereinthe amino acid positions of the polypeptide sequence are numbered withreference to SEQ ID NO: 546 or 1494. In some further embodiments, therecombinant amylase comprises a polypeptide sequence having at least80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or more sequence identity to SEQ ID NO: 546 or 1494, or afunctional fragment thereof, and wherein the recombinant amylasecomprises at least one substitution or substitution set selected from123R, 123R/126R/180E/214L/298Q/302W/393R, 123R/126R/180Q/214G/393R/428T,123R/126R/210P/298Q/302W/393R, 123R/126R/210P/428T, 123R/126R/428T,123R/210P/302R, 126R/180E/210P/214G/428T, 126R/180E/298Q/302R/393R,126R/180Q/210P/214L/298Q/302R, 126R/210P/302R/428T, 126R/214G/393R,126R/298Q/302H/393R, 126R/298Q/302R/393R/428T, 129W, 129W/228V/253R,131H/228V/279V, 131Y/282T, 180Q/214L, 210P/214L/302R/393R/428T,228V/282T/444R, 253R/279L/492G, 253R/279V/470S, 279L, 279L/470S, and441R, wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 546 or 1494. In some furtherembodiments, the recombinant amylase comprises a polypeptide sequencehaving at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 546 or1494, or a functional fragment thereof, and wherein the recombinantamylase comprises at least one substitution or substitution set selectedfrom D123R, D123R/T126R/D180E/E214L/H298Q/Q302W/E393R,D123R/T126R/D180Q/E214G/E393R/D428T,D123R/T126R/H210P/H298Q/Q302W/E393R, D123R/T126R/H210P/D428T,D123R/T126R/D428T, D123R/H210P/Q302R, T126R/D180E/H210P/E214G/D428T,T126R/D180E/H298Q/Q302R/E393R, T126R/D180Q/H210P/E214L/H298Q/Q302R,T126R/H210P/Q302R/D428T, T126R/E214G/E393R, T126R/H298Q/Q302H/E393R,T126R/H298Q/Q302R/E393R/D428T, D129W, D129W/Q228V/Q253R,E131H/Q228V/D279V, E131Y/E282T, D180Q/E214L,H210P/E214L/Q302R/E393R/D428T, Q228V/E282T/E444R, Q253R/D279L/E492G,Q253R/D279V/V470S, D279L, D279L/V470S, and D441R, wherein the amino acidpositions of the polypeptide sequence are numbered with reference to SEQID NO: 546 or 1494.

In some additional embodiments, the recombinant amylase comprises apolypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO: 632 or 1580, or a functional fragment thereof, and whereinthe recombinant amylase comprises at least one substitution orsubstitution set at one or more positions selected from 2, 45/166/298,48, 48/166/170/203, 52, 58/166, 58/203, 72, 82, 114, 114/223/306/308,149, 165, 166/203, 167, 170, 184/216/306/308, 184/223/306/437, 189, 203,225, 255, 258, 275, 280, 284, 294, 298, 396, 397, 411, 431, 448, 468,482, 483, and 495, wherein the amino acid positions of the polypeptidesequence are numbered with reference to SEQ ID NO: 632 or 1580. In somefurther embodiments, the recombinant amylase comprises a polypeptidesequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ IDNO: 632 or 1580, or a functional fragment thereof, and wherein therecombinant amylase comprises at least one substitution or substitutionset selected from 2L, 45S/166L/298D, 48D, 48D/166L/170S/203S, 52R,58A/166L, 58A/203S, 72T, 82A, 114R, 114R/223L/306F/308L, 149K, 165V,166L/203S, 167Y, 170S, 184D/216L/306F/308L, 184D/223L/306F/437L, 189D,203S, 225T, 255M, 255R, 258S, 275I, 275R, 280C, 284L, 284M, 294A, 298D,298N, 396E, 396Q, 397M, 411E, 431T, 448P, 468V, 482L, 483A, 495G, and495L, wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 632 or 1580. In some furtherembodiments, the recombinant amylase comprises a polypeptide sequencehaving at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 632 or1580, or a functional fragment thereof, and wherein the recombinantamylase comprises at least one substitution or substitution set selectedfrom R2L, Y45S/I166L/H298D, A48D, A48D/I166L/D170S/E203S, Y52R,T58A/I166L, T58A/E203S, S72T, Q82A, Hi 14R, H114R/V223L/Y306F/M308L,Q149K, H165V, I166L/E203S, E167Y, D170S, E184D/I216L/Y306F/M308L,E184D/V223L/Y306F/M437L, Q189D, E203S, E225T, V255M, V255R, K258S,Q275I, Q275R, Q280C, K284L, K284M, M294A, H298D, H298N, D396E, D396Q,E397M, T411E, P431T, H448P, R468V, N482L, G483A, I495G, and I495L,wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 632 or 1580.

In some additional embodiments, the recombinant amylase comprises apolypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO: 664 or 1612, or a functional fragment thereof, and whereinthe recombinant amylase comprises at least one substitution orsubstitution set at one or more positions selected from6/39/103/324/420, 41, 58/500/501, 61, 95/98/205/227/423/458,95/98/227/423, 95/205, 95/252/285/324, 123/170/197/275/298/396/448/483,123/197, 123/197/225/294/396/448/469, 123/284/294/324/396/448,134/225/275/302, 165/225/284/298/469/483, 170/275/294/298/448, 205/206,205/206/224/315/458/480, 206/227, 206/227/315/423, 225/294/298/396, 227,392/503, 458, 499, and 500, wherein the amino acid positions of thepolypeptide sequence are numbered with reference to SEQ ID NO: 664 or1612. In some further embodiments, the recombinant amylase comprises apolypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO: 664 or 1612, or a functional fragment thereof, and whereinthe recombinant amylase comprises at least one substitution orsubstitution set selected from 6I/39I/103D/324T/420V, 41F,58S/500P/501P, 61Y, 95L/252I/285V/324T, 95V/98I/205V/227V/423V/458F,95V/98I/227V/423V, 95V/205V, 123E/170S/197Y/275I/298N/396Q/448Q/483Q,123E/197Y, 123E/197Y/225G/294A/396Q/448Q/469I,123E/284L/294A/324T/396Q/448Q, 134R/225G/275I/302W,165V/225G/284L/298N/469I/483Q, 170S/275I/294A/298N/448Q, 205V/206E,205V/206E/224L/315S/458F/480L, 206E/227V, 206E/227V/315S/423V,225G/294A/298N/396Q, 227C, 392C/503L, 458F, 458Y, 499R, and 500P,wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 664 or 1612. In some furtherembodiments, the recombinant amylase comprises a polypeptide sequencehaving at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 664 or1612, or a functional fragment thereof, and wherein the recombinantamylase comprises at least one substitution or substitution set selectedfrom L61/V39I/V103D/A324T/I420V, L41F, T58S/H500P/H501P, L61Y,I95L/V252I/T285V/A324T, I95V/L98I/I205V/T227V/S423V/W458F,I95V/L98I/T227V/S423V, I95V/I205V,D123E/D170S/F197Y/Q275I/H298N/D396Q/H448Q/G483Q, D123E/F197Y,D123E/F197Y/E225G/M294A/D396Q/H448Q/V469I,D123E/K284L/M294A/A324T/D396Q/H448Q, E134R/E225G/Q275I/Q302W,H165V/E225G/K284L/H298N/V469I/G483Q, D170S/Q275I/M294A/H298N/H448Q,I205V/D206E, I205V/D206E/M224L/T315S/W458F/F480L, D206E/T227V,D206E/T227V/T315S/S423V, E225G/M294A/H298N/D396Q, T227C, I392C/H503L,W458F, W458Y, G499R, and H500P, wherein the amino acid positions of thepolypeptide sequence are numbered with reference to SEQ ID NO: 664 or1612.

In some additional embodiments, the recombinant amylase comprises apolypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO: 664 or 1612, or a functional fragment thereof, and whereinthe recombinant amylase comprises at least one substitution orsubstitution set at one or more positions selected from 95/252/285/324,123/197, 123/197/225/294/396/448/469, 134/225/275/302, 205/206, and205/206/224/315/458/480, wherein the amino acid positions of thepolypeptide sequence are numbered with reference to SEQ ID NO: 664 or1612. In some further embodiments, the recombinant amylase comprises apolypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO: 664 or 1612, or a functional fragment thereof, and whereinthe recombinant amylase comprises at least one substitution orsubstitution set selected from 95L/252I/285V/324T, 123E/197Y,123E/197Y/225G/294A/396Q/448Q/469I, 134R/225G/275I/302W, 205V/206E, and205V/206E/224L/315S/458F/480L, wherein the amino acid positions of thepolypeptide sequence are numbered with reference to SEQ ID NO: 664 or1612. In some further embodiments, the recombinant amylase comprises apolypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO: 664 or 1612, or a functional fragment thereof, and whereinthe recombinant amylase comprises at least one substitution orsubstitution set selected from I95L/V252I/T285V/A324T, D123E/F197Y,D123E/F197Y/E225G/M294A/D396Q/H448Q/V469I, E134R/E225G/Q275I/Q302W,I205V/D206E, and I205V/D206E/M224L/T315S/W458F/F480L, wherein the aminoacid positions of the polypeptide sequence are numbered with referenceto SEQ ID NO: 664 or 1612.

In some additional embodiments, the recombinant amylase comprises apolypeptide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO: 780 or 1728, or a functional fragment thereof, and whereinthe recombinant amylase comprises a substitution at position 126,wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 780 or 1728. In some furtherembodiments, the recombinant amylase comprises a polypeptide sequencehaving at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 780 or1728, or a functional fragment thereof, and wherein the recombinantamylase comprises at least one substitution or substitution set selectedfrom 126C, 126D, 126E, 126K, 126L, 126M, 126N, 126Q, 126S, 126T, and126W, wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 780 or 1728. In some furtherembodiments, the recombinant amylase comprises a polypeptide sequencehaving at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 780 or1728, or a functional fragment thereof, and wherein the recombinantamylase comprises at least one substitution or substitution set selectedfrom R126C, R126D, R126E, R126K, R126L, R126M, R126N, R126Q, R126S,R126T, and R126W, wherein the amino acid positions of the polypeptidesequence are numbered with reference to SEQ ID NO: 780 or 1728.

In some additional embodiments, the recombinant amylase comprises atleast one mutation in at least one position as provided in Table 4-1,4-2, 4-3, 4-4, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 4-11, 4-12, 4-13, and/or4-14, wherein the positions are numbered with reference to SEQ ID NO: 2,18, 40, 156, 174, 276, 308, 422, 456, 546, 632, 664, 780, 988, 1104,1122, 1224, 1256, 1370, 1404, 1494, 1580, 1612, and/or 1728. In somefurther embodiments, the recombinant amylase comprises a polypeptidesequence having at least about 80%, 85%, about 86%, about 87%, about88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%,about 95%, about 96%, about 97%, about 98%, about 99%, or more sequenceidentity to at least one sequence set forth in the even-numberedsequences of SEQ ID NOS: 4-18, 22-966, and 970-1914. In some furtherembodiments, the recombinant amylase comprises a polypeptide sequencehaving at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more sequence identity to at least onesequence set forth in the even-numbered sequences of SEQ ID NOS: 4-18,22-966, and 970-1914. In some additional embodiments, the recombinantamylase comprises at least one sequence set forth in the even-numberedsequences of SEQ ID NOS: 4-18, 22-966, and 970-1914.

In some embodiments, the recombinant amylase comprises at least onesequence set forth in the even-numbered sequences of SEQ ID NOS: 22-124and 970-1072. In some embodiments, the recombinant amylase comprises atleast one sequence set forth in the even-numbered sequences of SEQ IDNOS: 126-172 and 1074-1120. In some embodiments, the recombinant amylasecomprises at least one sequence set forth in the even-numbered sequencesof SEQ ID NOS: 174-222 and 1122-1170. In some embodiments, therecombinant amylase comprises at least one sequence set forth in theeven-numbered sequences of SEQ ID NOS: 224-306 and 1172-1254. In someembodiments, the recombinant amylase comprises at least one sequence setforth in the even-numbered sequences of SEQ ID NOS: 308-380 and1256-1328. In some embodiments, the recombinant amylase comprises atleast one sequence set forth in the even-numbered sequences of SEQ IDNOS: 382-440 and 1330-1388. In some embodiments, the recombinant amylasecomprises at least one sequence set forth in the even-numbered sequencesof SEQ ID NOS: 442-540 and 1390-1488. In some embodiments, therecombinant amylase comprises at least one sequence set forth in theeven-numbered sequences of SEQ ID NOS: 542-608 and 1490-1556. In someembodiments, the recombinant amylase comprises at least one sequence setforth in the even-numbered sequences of SEQ ID NOS: 610-660 and1558-1608. In some embodiments, the recombinant amylase comprises atleast one sequence set forth in the even-numbered sequences of SEQ IDNOS: 662-746 and 1610-1694. In some embodiments, the recombinant amylasecomprises at least one sequence set forth in the even-numbered sequencesof SEQ ID NOS: 748-798 and 1696-1746. In some embodiments, therecombinant amylase comprises at least one sequence set forth in theeven-numbered sequences of SEQ ID NOS: 800-820 and 1748-1768. In someembodiments, the recombinant amylase comprises at least one sequence setforth in the even-numbered sequences of SEQ ID NOS: 822-966 and1770-1914.

In some embodiments, the engineered amylase polypeptide comprises afunctional fragment of an engineered amylase polypeptide encompassed bythe invention. Functional fragments have at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% of the activity of the engineered amylase polypeptide fromwhich is was derived (i.e., the parent engineered amylase). In someembodiments, a functional fragment comprises at least 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% and even 99% of the parent sequence of the engineered amylase.In some embodiments the functional fragment is truncated by less than 5,less than 10, less than 15, less than 10, less than 25, less than 30,less than 35, less than 40, less than 45, and less than 50 amino acids.

In some additional embodiments, the recombinant amylase retains moreenzymatic activity after exposure to high and/or low temperatures, ascompared to a reference sequence. In some embodiments, the referencesequence is wild-type amylase, while in some other embodiments, thereference sequence is another recombinant amylase. In some additionalembodiments, the recombinant amylase is more thermostable than theamylase of SEQ ID NO: 2. In some further embodiments, the recombinantamylase is more thermostable than the amylase of SEQ ID NO: 18, 40, 156,174, 276, 308, 422, 456, 546, 632, 664, 780, 988, 1104, 1122, 1224,1256, 1370, 1404, 1494, 1580, 1612, and/or 1728. In some embodiments,the recombinant amylase is stable at 30° C., 37° C., 40° C., 50° C., 60°C., and/or 95° C., and/or 40° C. to 60° C. In some embodiments, therecombinant amylase is stable at 30° C., 37° C., 42° C., and/or 48° C.In some additional embodiments, the recombinant amylase is more stablethan a reference sequence at 30° C., 37° C., 40° C., 50° C., 60° C.,and/or 95° C., and/or 40° C. to 60° C. In some embodiments, therecombinant amylase is more stable than a reference sequence at 30° C.,37° C., 40° C., 50° C., 60° C., and/or 95° C., and/or 40° C. to 60° C.In some additional embodiments, the recombinant amylase is morethermostable than the amylase of SEQ ID NO: 2. In some furtherembodiments, the recombinant amylase is more thermostable than theamylase of SEQ ID NO: 18, 40, 156, 174, 276, 308, 422, 456, 546, 632,664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494, 1580, 1612,and/or 1728.

In some embodiments, the recombinant amylase is stable in low pHenvironments, while in other embodiments, the recombinant amylase isstable in high pH environments, and in still further embodiments, therecombinant is stable in neutral pH environments. In some embodiments,the recombinant amylase is stable in low, high, and/or neutral pHenvironments. In some embodiments, the recombinant amylase retainsenzymatic activity after exposure to a low, high, and/or neutral pHenvironment. In some additional embodiments, the recombinant amylase ismore stable in high, low, and/or neutral pH environment(s), as comparedto a reference sequence. In some embodiments, the reference sequence iswild-type amylase, while in other embodiments, the reference sequence isanother engineered amylase. In some additional embodiments, therecombinant amylase is more stable than the amylase of SEQ ID NO: 2. Insome further embodiments, the recombinant amylase is more stable thanthe amylase of SEQ ID NO: 18, 40, 156, 174, 276, 308, 422, 456, 546,632, 664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494, 1580,1612, and/or 1728. In some additional embodiments, the recombinantamylase is more stable at pHs greater than 7, than the amylase of SEQ IDNO: 2. In some additional embodiments, the recombinant amylase is morestable at pHs greater than 7, than the amylase of SEQ ID NO: 18, 40,156, 174, 276, 308, 422, 456, 546, 632, 664, 780, 988, 1104, 1122, 1224,1256, 1370, 1404, 1494, 1580, 1612, and/or 1728. In some additionalembodiments, the recombinant amylase is more stable at pH 7.8, than theamylase of SEQ ID NO: 2. In some additional embodiments, the recombinantamylase is more stable at pH 7.8, than the amylase of SEQ ID NO: 18, 40,156, 174, 276, 308, 422, 456, 546, 632, 664, 780, 988, 1104, 1122, 1224,1256, 1370, 1404, 1494, 1580, 1612, and/or 1728. In some additionalembodiments, the recombinant amylase is more stable at pH 7.5, than theamylase of SEQ ID NO: 2. In some additional embodiments, the recombinantamylase is more stable at pH 7.5, than the amylase of SEQ ID NO: 18, 40,156, 174, 276, 308, 422, 456, 546, 632, 664, 780, 988, 1104, 1122, 1224,1256, 1370, 1404, 1494, 1580, 1612, and/or 1728. In some additionalembodiments, the recombinant amylase is more stable at pH 7, than theamylase of SEQ ID NO: 2. In some additional embodiments, the recombinantamylase is more stable at pH 7, than the amylase of SEQ ID NO: 18, 40,156, 174, 276, 308, 422, 456, 546, 632, 664, 780, 988, 1104, 1122, 1224,1256, 1370, 1404, 1494, 1580, 1612, and/or 1728. In some furtherembodiments, the recombinant amylase is more stable at pHs less than 7,than the amylase of SEQ ID NO: 2. In some additional embodiments, therecombinant amylase is more stable at pHs less than pH 7, than theamylase of SEQ ID NO: 18, 40, 156, 174, 276, 308, 422, 456, 546, 632,664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494, 1580, 1612,and/or 1728. In some additional embodiments, the recombinant amylase ismore stable at pH 6.8, than the amylase of SEQ ID NO: 2. In someadditional embodiments, the recombinant amylase is more stable at pH6.8, than the amylase of SEQ ID NO: 18, 40, 156, 174, 276, 308, 422,456, 546, 632, 664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494,1580, 1612, and/or 1728. In some additional embodiments, the recombinantamylase is more stable at pH 6.7, than the amylase of SEQ ID NO: 2. Insome additional embodiments, the recombinant amylase is more stable atpH 6.7, than the amylase of SEQ ID NO: 18, 40, 156, 174, 276, 308, 422,456, 546, 632, 664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494,1580, 1612, and/or 1728. In some additional embodiments, the recombinantamylase is more stable at pH 6.5, than the amylase of SEQ ID NO: 2. Insome additional embodiments, the recombinant amylase is more stable atpH 6.5, than the amylase of SEQ ID NO: 18, 40, 156, 174, 276, 308, 422,456, 546, 632, 664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494,1580, 1612, and/or 1728. In some additional embodiments, the recombinantamylase is more stable at pH 6, than the amylase of SEQ ID NO: 2. Insome additional embodiments, the recombinant amylase is more stable atpH 6, than the amylase of SEQ ID NO: 18, 40, 156, 174, 276, 308, 422,456, 546, 632, 664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494,1580, 1612, and/or 1728. In some further embodiments, the recombinantamylase is more stable at pHs less than 5, than the amylase of SEQ IDNO: 2. In some additional embodiments, the recombinant amylase is morestable at pH 5, than the amylase of SEQ ID NO: 2. In some additionalembodiments, the recombinant amylase is more stable at pH 5, than theamylase of SEQ ID NO: 18, 40, 156, 174, 276, 308, 422, 456, 546, 632,664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494, 1580, 1612,and/or 1728. In some further embodiments, the recombinant amylase ismore stable at pH 4, than the amylase of SEQ ID NO: 2. In some furtherembodiments, the recombinant amylase is more stable at pH 5, than theamylase of SEQ ID NO: 18, 40, 156, 174, 276, 308, 422, 456, 546, 632,664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494, 1580, 1612,and/or 1728. In some additional embodiments, the recombinant amylase ismore stable at pH 3.8, than the amylase of SEQ ID NO: 2. In someadditional embodiments, the recombinant amylase is more stable at pH3.8, than the amylase of SEQ ID NO: 18, 40, 156, 174, 276, 308, 422,456, 546, 632, 664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494,1580, 1612, and/or 1728.

In some additional embodiments, the recombinant amylase is more stableat pH 3.5, than the amylase of SEQ ID NO: 2. In some additionalembodiments, the recombinant amylase is more stable at pH 3.5, than theamylase of SEQ ID NO: 18, 40, 156, 174, 276, 308, 422, 456, 546, 632,664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494, 1580, 1612,and/or 1728. In some additional embodiments, the recombinant amylase ismore stable at pH 3.25, than the amylase of SEQ ID NO: 2. In someadditional embodiments, the recombinant amylase is more stable at pH3.25, than the amylase of SEQ ID NO: 18, 40, 156, 174, 276, 308, 422,456, 546, 632, 664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494,1580, 1612, and/or 1728. In some further embodiments, the recombinantamylase is more stable at pH 3, than the amylase of SEQ ID NO: 2. Insome further embodiments, the recombinant amylase is more stable at pH3, than the amylase of SEQ ID NO: 18, 40, 156, 174, 276, 308, 422, 456,546, 632, 664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494, 1580,1612, and/or 1728. In some additional embodiments, the recombinantamylase is more stable at pH 2.75, than the amylase of SEQ ID NO: 2. Insome additional embodiments, the recombinant amylase is more stable atpH 2.75, than the amylase of SEQ ID NO: 18, 40, 156, 174, 276, 308, 422,456, 546, 632, 664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494,1580, 1612, and/or 1728. In some further embodiments, the recombinantamylase is more stable at pH 2.5, than the amylase of SEQ ID NO: 2. Insome further embodiments, the recombinant amylase is more stable at pH2.5, than the amylase of SEQ ID NO: 18, 40, 156, 174, 276, 308, 422,456, 546, 632, 664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494,1580, 1612, and/or 1728. In some further embodiments, the recombinantamylase is more stable at pH 2.3, than the amylase of SEQ ID NO: 2. Insome further embodiments, the recombinant amylase is more stable at pH2.3, than the amylase of SEQ ID NO: 18, 40, 156, 174, 276, 308, 422,456, 546, 632, 664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494,1580, 1612, and/or 1728. In some further embodiments, the recombinantamylase is more stable at pH 2, than the amylase of SEQ ID NO: 2. Insome further embodiments, the recombinant amylase is more stable at pH2, than the amylase of SEQ ID NO: 18, 40, 156, 174, 276, 308, 422, 456,546, 632, 664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494, 1580,1612, and/or 1728.

In some embodiments, the recombinant amylase is more resistant toproteolysis than the amylase of SEQ ID NO: 2. In some embodiments, therecombinant amylase is more resistant to proteolysis than the amylase ofSEQ ID NO: 18, 40, 156, 174, 276, 308, 422, 456, 546, 632, 664, 780,988, 1104, 1122, 1224, 1256, 1370, 1404, 1494, 1580, 1612, and/or 1728.In some embodiments, the recombinant amylase is resistant to proteolysisby pepsin. In some additional embodiments, the recombinant amylase ismore resistant to proteolysis by pepsin than the amylase of SEQ ID NO:2. In some embodiments, the recombinant amylase is more resistant toproteolysis by pepsin than the amylase of SEQ ID NO: 18, 40, 156, 174,276, 308, 422, 456, 546, 632, 664, 780, 988, 1104, 1122, 1224, 1256,1370, 1404, 1494, 1580, 1612, and/or 1728. In some additionalembodiments, the recombinant amylase is resistant to proteolysis bytrypsin. In some additional embodiments, the recombinant amylase is moreresistant to proteolysis by trypsin than the amylase of SEQ ID NO: 2. Insome embodiments, the recombinant amylase is more resistant toproteolysis by trypsin than the amylase of SEQ ID NO: 18, 40, 156, 174,276, 308, 422, 456, 546, 632, 664, 780, 988, 1104, 1122, 1224, 1256,1370, 1404, 1494, 1580, 1612, and/or 1728. In some embodiments, therecombinant amylase is resistant to proteolysis by chymotrypsin. In someadditional embodiments, the recombinant amylase is more resistant toproteolysis by chymotrypsin than the amylase of SEQ ID NO: 2. In someembodiments, the recombinant amylase is more resistant to proteolysis bychymotrypsin than the amylase of SEQ ID NO: 18, 40, 156, 174, 276, 308,422, 456, 546, 632, 664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404,1494, 1580, 1612, and/or 1728. In some further embodiments, therecombinant amylase is resistant to proteolysis by pepsin, trypsin,and/or chymotrypsin. In some additional embodiments, the recombinantamylase is more resistant to proteolysis by pepsin, trypsin, and/orchymotrypsin, than the amylase of SEQ ID NO: 2. In some embodiments, therecombinant amylase is more resistant to proteolysis by pepsin, trypsin,and/or chymotrypsin, than the amylase of SEQ ID NO: 18, 40, 156, 174,276, 308, 422, 456, 546, 632, 664, 780, 988, 1104, 1122, 1224, 1256,1370, 1404, 1494, 1580, 1612, and/or 1728.

In some additional embodiments, the recombinant amylase is active in thepresence of at least one bile salt. In some additional embodiments, therecombinant amylase is more active in the presence of at least one bilesalt than the amylase of SEQ ID NO: 2. In some additional embodiments,the recombinant amylase is more active in the presence of at least onebile salt than the amylase of SEQ ID NO: 18, 40, 156, 174, 276, 308,422, 456, 546, 632, 664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404,1494, 1580, 1612, and/or 1728. In some embodiments, the recombinantamylase retains enzymatic activity after exposure to a bile salt. Insome additional embodiments, the recombinant amylase retains moreenzymatic activity after exposure to a bile salt, as compared to areference sequence. In some embodiments, the reference sequence is theamylase of SEQ ID NO: 2, 18, 40, 156, 174, 276, 308, 422, 456, 546, 632,664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494, 1580, 1612,and/or 1728. In some additional embodiments, the bile salt istaurocholate.

In yet some additional embodiments, the recombinant amylase exhibitsmore than one improved property, as compared to wild-type amylase oranother recombinant amylase. In some embodiments, the recombinantamylase exhibits more than one improved property as compared to SEQ IDNO:2, while in some additional embodiments, the recombinant amylaseexhibits more than one improved property as compared to SEQ ID NO: 18,40, 156, 174, 276, 308, 422, 456, 546, 632, 664, 780, 988, 1104, 1122,1224, 1256, 1370, 1404, 1494, 1580, 1612, and/or 1728. In someembodiments, the improved properties are selected from acid stability,alkaline stability, stability and/or activity at acidic pH, stability atalkaline pH, stability at neutral pH, thermostability, proteolysisresistance, and increased activity in the presence of at least one bilesalt. In yet some additional embodiments, the recombinant amylase ismore stable and/or active at acidic pHs, more thermostable, moreresistant to proteolysis, and/or more active in the presence of at leastone bile salt than the amylase of SEQ ID NO: 2, 18, 40, 156, 174, 276,308, 422, 456, 546, 632, 664, 780, 988, 1104, 1122, 1224, 1256, 1370,1404, 1494, 1580, 1612, and/or 1728. It is contemplated that anycombination of improved properties will find use in the presentinvention. It is not intended that the present invention be limited toany specific combination of improved properties. Furthermore, in someembodiments, there are two improved properties, while in some otherembodiments, there are three improved properties, in some additionalembodiments, there are four improved properties, and in some additionalembodiments, there are five or more improved properties. It is alsocontemplated that the recombinant amylase of the present inventionfurthers comprise additional improvements. In some embodiments, theseadditional improvements provide advantages over wild-type amylase, whilein some other embodiments, the additional improvements will provideadvantages over other recombinant amylases.

In some embodiments, the recombinant amylase exhibits at least oneimproved property selected from improved stability and/or activity atacidic pHs, improved thermostability, improved resistance toproteolysis, and/or improved activity in the presence of at least onebile salt, as compared to the amylase of SEQ ID NO: 2. In someembodiments, the recombinant amylase exhibits at least one improvedproperty selected from improved stability and/or activity at acidic pHs,improved thermostability, improved resistance to proteolysis, and/orimproved activity in the presence of at least one bile salt, as comparedto the amylase of SEQ ID NO: 18, 40, 156, 174, 276, 308, 422, 456, 546,632, 664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494, 1580,1612, and/or 1728. In some embodiments, the recombinant amylase exhibitsat least two improved properties selected from improved stability and/oractivity at acidic pHs, improved thermostability, improved resistance toproteolysis, and/or improved activity in the presence of at least onebile salt, as compared to the amylase of SEQ ID NO: 2. In someembodiments, the recombinant amylase exhibits at least two improvedproperties selected from improved stability and/or activity at acidicpHs, improved thermostability, improved resistance to proteolysis,and/or improved activity in the presence of at least one bile salt, ascompared to the amylase of SEQ ID NO: 18, 40, 156, 174, 276, 308, 422,456, 546, 632, 664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494,1580, 1612, and/or 1728. In some embodiments, the recombinant amylaseexhibits at least three improved properties selected from improvedstability and/or activity at acidic pHs, improved thermostability,improved resistance to proteolysis, and/or improved activity in thepresence of at least one bile salt, as compared to the amylase of SEQ IDNO: 2. In some embodiments, the recombinant amylase exhibits at leastthree improved properties selected from improved stability and/oractivity at acidic pHs, improved thermostability, improved resistance toproteolysis, and/or improved activity in the presence of at least onebile salt, as compared to the amylase of SEQ ID NO: 18, 40, 156, 174,276, 308, 422, 456, 546, 632, 664, 780, 988, 1104, 1122, 1224, 1256,1370, 1404, 1494, 1580, 1612, and/or 1728. In some embodiments, therecombinant amylase exhibits the improved properties of improvedstability and/or activity at acidic pHs, improved thermostability,improved resistance to proteolysis, and improved activity in thepresence of at least one bile salt, as compared to the amylase of SEQ IDNO: 2. In some embodiments, the recombinant amylase exhibits theimproved properties of improved stability and/or activity at acidic pHs,improved thermostability, improved resistance to proteolysis, and/orimproved activity in the presence of at least one bile salt, as comparedto the amylase of SEQ ID NO: 18, 40, 156, 174, 276, 308, 422, 456, 546,632, 664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494, 1580,1612, and/or 1728. In some embodiments, the recombinant amylase exhibitsat least one improved property selected from improved stability and/oractivity at acidic pHs, improved thermostability, improved resistance toproteolysis, and/or improved activity in the presence of at least onebile salt, as well as at least one additional improved property, ascompared to the amylase of SEQ ID NO: 2. In some embodiments, therecombinant amylase exhibits at least one improved property selectedfrom improved stability and/or activity at acidic pHs, improvedthermostability, improved resistance to proteolysis, and/or improvedactivity in the presence of at least one bile salt, as well as at leastone additional improved property, as compared to the amylase of SEQ IDNO: 18, 40, 156, 174, 276, 308, 422, 456, 546, 632, 664, 780, 988, 1104,1122, 1224, 1256, 1370, 1404, 1494, 1580, 1612, and/or 1728. In someembodiments, the recombinant amylase exhibits at least one improvedproperty selected from: i) enhanced catalytic activity; ii) increasedtolerance to acid pH; iii) increased tolerance to pH 2.3, 2.5, 2.75, 3,3.25, and/or 3.5; iv) increased tolerance to pH 6.7 and/or 6.8; v)increased tolerance to at least one protease; vi) increased tolerance toat least one bile salt; vii) increased thermotolerance; or a combinationof any of i), ii), iii), iv), v), vi), and vii) as compared to areference sequence. In some embodiments, the reference sequence is SEQID NO: 2, while in some alternative embodiments, the reference sequenceis selected from SEQ ID NO: 18, 40, 156, 174, 276, 308, 422, 456, 546,632, 664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494, 1580,1612, and/or 1728. In some embodiments, the recombinant amylase exhibitsat least one improved property selected from: i) enhanced catalyticactivity; ii) increased tolerance to acid pH; iii) increased toleranceto pH 2.3, 2.5, 2.75, 3, 3.25, and/or 3.5; iv) increased tolerance to pH6.7 and/or 6.8; v) increased tolerance to at least one protease; vi)increased tolerance to at least one bile salt; vii) increasedthermotolerance; or a combination of any of i), ii), iii), iv), v), vi),and vii) as compared to at least one reference sequence. In someembodiments, the reference sequence is SEQ ID NO: 2, while in somealternative embodiments, the reference sequence is selected from SEQ IDNO: 18, 40, 156, 174, 276, 308, 422, 456, 546, 632, 664, 780, 988, 1104,1122, 1224, 1256, 1370, 1404, 1494, 1580, 1612, and/or 1728. In someembodiments, the recombinant amylase exhibits at least one improvedproperty selected from: i) enhanced catalytic activity; ii) increasedtolerance to acid pH; iii) increased tolerance to pH 2.3, 2.5, 2.75, 3,3.25, and/or 3.5; iv) increased tolerance to pH 6.7 and/or 6.8; v)increased tolerance to at least one protease; vi) increased tolerance toat least one bile salt; vii) increased thermotolerance; or a combinationof any of i), ii), iii), iv), v), vi), and vii) as compared to at leasttwo or more reference sequences. In some embodiments, the referencesequence is selected from SEQ ID NO: 18, 40, 156, 174, 276, 308, 422,456, 546, 632, 664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494,1580, 1612, and/or 1728. In some further embodiments, the recombinantamylase is purified.

The present invention further provides methods starch hydrolysis,comprising providing starch and at least one engineered amylase providedherein; and exposing the starch to the engineered amylase underconditions such that the starch is hydrolyzed by said engineeredamylase.

In still some further embodiments, the amylase is stable in food and/orbeverages. In some additional embodiments, the amylase is stable innutritional and other supplements. In some embodiments, the supplementsare liquid, while in other embodiments, they are emulsions, suspensions,or solids. It is not intended that the present invention be limited toany particular food, beverage, and/or supplement format or form.

Polynucleotides Encoding Engineered Polypeptides, Expression Vectors andHost Cells

The present invention provides polynucleotides encoding the engineeredamylase polypeptides described herein. In some embodiments, thepolynucleotides are operatively linked to one or more heterologousregulatory sequences that control gene expression to create arecombinant polynucleotide capable of expressing the polypeptide.Expression constructs containing a heterologous polynucleotide encodingthe engineered amylase polypeptides can be introduced into appropriatehost cells to express the corresponding amylase polypeptide.

As will be apparent to the skilled artisan, availability of a proteinsequence and the knowledge of the codons corresponding to the variousamino acids provide a description of all the polynucleotides capable ofencoding the subject polypeptides. The degeneracy of the genetic code,where the same amino acids are encoded by alternative or synonymouscodons, allows an extremely large number of nucleic acids to be made,all of which encode the engineered amylase polypeptide. Thus, havingknowledge of a particular amino acid sequence, those skilled in the artcould make any number of different nucleic acids by simply modifying thesequence of one or more codons in a way which does not change the aminoacid sequence of the protein. In this regard, the present inventionspecifically contemplates each and every possible variation ofpolynucleotides that could be made encoding the polypeptides describedherein by selecting combinations based on the possible codon choices,and all such variations are to be considered specifically disclosed forany polypeptide described herein, including the variants provided inTable 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 4-11, 4-12,4-13, and/or 4-14; and variants provided in Table 4-1, 4-2, 4-3, 4-4,4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 4-11, 4-12, 4-13, and/or 4-14, where thevariants are absent the histidine tag and preceding amino acid linker.

In various embodiments, the codons are preferably selected to fit thehost cell in which the protein is being produced. For example, preferredcodons used in bacteria are used for expression in bacteria, whilepreferred codons used in fungi are used for expression in fungi.Consequently, codon optimized polynucleotides encoding the engineeredamylase polypeptides contain preferred codons at about 40%, 50%, 60%,70%, 80%, or greater than 90% of codon positions of the full lengthcoding region.

In some embodiments, the present invention provides a recombinantpolynucleotide sequence having at least about 80%, at least about 85%,about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,about 99%, or more sequence identity to SEQ ID NO: 1, 17, 39, 155, 173,275, 307, 421, 455, 545, 631, 663, 779, 987, 1103, 1121, 1223, 1255,1369, 1403, 1493, 1579, 1611, and/or 1727. In some embodiments, thepresent invention provides a recombinant polynucleotide sequence havingat least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 1, 17, 39,155, 173, 275, 307, 421, 455, 545, 631, 663, 779, 987, 1103, 1121, 1223,1255, 1369, 1403, 1493, 1579, 1611, and/or 1727. In some embodiments,the recombinant polynucleotide sequence has at least 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity to the odd-numbered sequences of SEQ ID NOS: 3-17,21-965, and 969-1913. In some embodiments, the polynucleotide encodingthe engineered amylase polypeptides comprises the polynucleotidesequence of SEQ ID NO: 1, 17, 39, 155, 173, 275, 307, 421, 455, 545,631, 663, 779, 987, 1103, 1121, 1223, 1255, 1369, 1403, 1493, 1579,1611, or 1727. In some embodiments, the polynucleotide encoding theengineered amylase polypeptides comprises the polynucleotide sequence ofodd-numbered sequences of SEQ ID NOS: 3-17, 21-965, and 969-1913.

In some embodiments, as described herein, the polynucleotide encodes anengineered polypeptide having amylase activity with the propertiesdisclosed herein, wherein the polypeptide comprises an amino acidsequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identityto: a reference sequence (e.g., SEQ ID NO: 2, 18, 40, 156, 174, 276,308, 422, 456, 546, 632, 664, 780, 988, 1104, 1122, 1224, 1256, 1370,1404, 1494, 1580, 1612, and/or 1728), or the amino acid sequence of anyvariant as disclosed in Table 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7, 4-8,4-9, 4-10, 4-11, 4-12, 4-13, and/or 4-14; or the amino acid sequence ofany variant as disclosed in Table 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7,4-8, 4-9, 4-10, 4-11, 4-12, 4-13, and/or 4-14, wherein the variant islacking the histidine tag and preceding amino acid linker, and one ormore residue differences as compared to the reference polypeptide of SEQID NO: 2, 18, 40, 156, 174, 276, 308, 422, 456, 546, 632, 664, 780, 988,1104, 1122, 1224, 1256, 1370, 1404, 1494, 1580, 1612, and/or 1728; orthe amino acid sequence of any variant as disclosed in Table 4-1, 4-2,4-3, 4-4, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 4-11, 4-12, 4-13, and/or 4-14,or the amino acid sequence of any variant as disclosed in the Tables(for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acidresidue positions), with or without a histidine tag and preceding aminoacid linker. In some embodiments, the polynucleotide encodes anengineered polypeptide having amylase activity with the propertiesdisclosed herein, wherein the polypeptide comprises an amino acidsequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity to reference sequence SEQ ID NO: 2, 18, 40, 156, 174, 276, 308,422, 456, 546, 632, 664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404,1494, 1580, 1612, and/or 1728, and one or more residue differences ascompared to SEQ ID NO: 2, 18, 40, 156, 174, 276, 308, 422, 456, 546,632, 664, 780, 988, 1104, 1122, 1224, 1256, 1370, 1404, 1494, 1580,1612, and/or 1728, at residue positions selected from those provided inTable 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 4-11, 4-12,4-13, and/or 4-14, when optimally aligned with the polypeptide of SEQ IDNO: 2, 18, 40, 156, 174, 276, 308, 422, 456, 546, 632, 664, 780, 988,1104, 1122, 1224, 1256, 1370, 1404, 1494, 1580, 1612, and/or 1728.

In some embodiments, the polynucleotide encodes a recombinant amylasecomprising a polypeptide sequence having at least 70%, 75%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ormore sequence identity to SEQ ID NO: 2 or 18, or a functional fragmentthereof.

In some embodiments, the polynucleotide encodes a recombinant amylasecomprising a polypeptide sequence having at least 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NO: 2 or 18, or a functional fragmentthereof, and wherein the encoded recombinant amylase comprises at leastone substitution or substitution set at one or more positions selectedfrom 2/73/256/493, 16/33/73/130/256/282/371/374/493,16/33/130/256/302/371/383/470, 16/33/157/256/302/371/383,16/73/74/256/282/366/371/383/493, 16/73/256/282/302/331/371/383,16/130/256/276/374/470, 16/256/302/374, 19/72/345/473,26/27/396/412/428/473, 26/27/412, 26/27/466/473, 29,73/130/256/282/302/493, 74/371/374/383/493, 130/256/493, 149, 158, 254,256, 256/383, 257, 259, 272, 279, 284, 317/383, 322, 345/388/396/428,388/396/412/428, 396/412, 396/412/428/466, 409, 412, and 495, whereinthe amino acid positions of the polypeptide sequence are numbered withreference to SEQ ID NO: 2 or 18. In some embodiments, the encodedrecombinant amylase comprises at least one substitution or substitutionset selected from 2K/73V/256S/493D,16D/33E/73V/130D/256S/282D/371F/374S/493D,16D/33E/130D/256S/302Q/371F/383E/470T, 16D/33E/157Y/256S/302Q/371F/383E,16D/73V/74A/256S/282D/366L/371F/383E/493D,16D/73V/256S/282D/302Q/331P/371F/383E, 16D/130D/256S/276Q/374S/470T,16D/256S/302Q/374S, 19E/72T/345A/473D, 26A/27L/396H/412D/428E/473D,26A/27L/412D, 26A/27L/466E/473D, 29G, 29R, 29S,73V/130D/256S/282D/302Q/493D, 74A/371F/374S/383E/493D, 130D/256S/493D,149R, 149T, 158R, 254G, 254S, 256S, 256S/383E, 257E, 257G, 257R, 257S,257V, 259Q, 272G, 272K, 272P, 272R, 272S, 279R, 284C, 284R, 284S, 284Y,317G/383L, 322A, 345A/388E/396H/428E, 388E/396H/412D/428E, 396H/412D,396H/412D/428E/466E, 409S, 412S, 495E, 495L, and 495Y, wherein the aminoacid positions of the polypeptide sequence are numbered with referenceto SEQ ID NO: 2 or 18. In some embodiments, the encoded recombinantamylase comprises at least one substitution or substitution set selectedfrom R2K/I73V/A256S/E493D,E16D/D33E/I73V/E130D/A256S/E282D/Y371F/H374S/E493D,E16D/D33E/E130D/A256S/M302Q/Y371F/Q383E/V470T,E16D/D33E/F157Y/A256S/M302Q/Y371F/Q383E,E16D/I73V/P74A/A256S/E282D/F366L/Y371F/Q383E/E493D,E16D/I73V/A256S/E282D/M302Q/H331P/Y371F/Q383E,E16D/E130D/A256S/T276Q/H374S/V470T, E16D/A256S/M302Q/H374S,K19E/S72T/P345A/E473D, E26A/R27L/D396H/L412D/D428E/E473D,E26A/R27L/L412D, E26A/R27L/Q466E/E473D, D29G, D29R, D29S,I73V/E130D/A256S/E282D/M302Q/E493D, P74A/Y371F/H374S/Q383E/E493D,E130D/A256S/E493D, Q149R, Q149T, K158R, E254G, E254S, A256S,A256S/Q383E, P257E, P257G, P257R, P257S, P257V, P259Q, D272G, D272K,D272P, D272R, D272S, D279R, K284C, K284R, K284S, K284Y, V317G/Q383L,F322A, P345A/D388E/D396H/D428E, D388E/D396H/L412D/D428E, D396H/L412D,D396H/L412D/D428E/Q466E, V409S, L412S, 1495E, I495L, and I495Y, whereinthe amino acid positions of the polypeptide sequence are numbered withreference to SEQ ID NO: 2 or 18.

In some embodiments, the polynucleotide encodes a recombinant amylasecomprising a polypeptide sequence having at least 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NO: 40 or 988, or a functional fragmentthereof, and wherein the encoded recombinant amylase comprises at leastone substitution or substitution set at one or more positions selectedfrom 26/29/158/272/412, 26/29/272/279/412/493, 26/29/272/396/412,26/29/279/412/428, 26/158/272/279/388/412/428, 26/158/272/412,26/272/279/412/428/493, 29/272/279/396/412, 130/149/254/284/322/409/495,130/149/257/284/322/409/466/473, 130/254/284/322/374/409/466,130/257/284/374/409/466, 130/284/322/374/409/473,149/257/284/322/409/466, 158/272/279/394/396/412/428/493,158/272/388/412/428/493, 158/272/412/428, 158/279/388/396/412/428,254/257/284/322/409/473, 254/284/409/466/495, 272/279/412/428/493,272/412/428, 272/412/493, and 284/322/409/473, wherein the amino acidpositions of the polypeptide sequence are numbered with reference to SEQID NO: 40 or 988. In some embodiments, the encoded recombinant amylasecomprises at least one substitution or substitution set selected from26A/29R/158R/272G/412D, 26A/29R/272G/279R/412D/493D,26A/29R/272G/396H/412D, 26A/29R/279R/412D/428E,26A/158R/272G/279R/388E/412D/428E, 26A/158R/272G/412D,26A/272G/279R/412D/428E/493D, 29R/272G/279R/396H/412D,130D/149R/254S/284S/322A/409S/495E,130D/149R/257G/284S/322A/409S/466E/473D,130D/254S/284S/322A/374S/409S/466E, 130D/257G/284S/374S/409S/466E,130D/284S/322A/374S/409S/473D, 149R/257G/284S/322A/409S/466E,158R/272G/279R/394R/396H/412D/428E/493D, 158R/272G/388E/412D/428E/493D,158R/272G/412D/428E, 158R/279R/388E/396H/412D/428E,254S/257G/284S/322A/409S/473D, 254S/284S/409S/466E/495E,272G/279R/412D/428E/493D, 272G/412D/428E, 272G/412D/493D, and284S/322A/409S/473D, wherein the amino acid positions of the polypeptidesequence are numbered with reference to SEQ ID NO: 40 or 988. In someembodiments, the encoded recombinant amylase comprises at least onesubstitution or substitution set selected fromE26A/D29R/K158R/D272G/L412D, E26A/D29R/D272G/D279R/L412D/E493D,E26A/D29R/D272G/D396H/L412D, E26A/D29R/D279R/L412D/D428E,E26A/K158R/D272G/D279R/D388E/L412D/D428E, E26A/K158R/D272G/L412D,E26A/D272G/D279R/L412D/D428E/E493D, D29R/D272G/D279R/D396H/L412D,E130D/Q149R/E254S/K284S/F322A/V409S/I495E,E130D/Q149R/P257G/K284S/F322A/V409S/Q466E/E473D,E130D/E254S/K284S/F322A/H374S/V409S/Q466E,E130D/P257G/K284S/H374S/V409S/Q466E,E130D/K284S/F322A/H374S/V409S/E473D,Q149R/P257G/K284S/F322A/V409S/Q466E,K158R/D272G/D279R/Q394R/D396H/L412D/D428E/E493D,K158R/D272G/D388E/L412D/D428E/E493D, K158R/D272G/L412D/D428E,K158R/D279R/D388E/D396H/L412D/D428E,E254S/P257G/K284S/F322A/V409S/E473D, E254S/K284S/V409S/Q466E/1495E,D272G/D279R/L412D/D428E/E493D, D272G/L412D/D428E, D272G/L412D/E493D, andK284S/F322A/V409S/E473D, wherein the amino acid positions of thepolypeptide sequence are numbered with reference to SEQ ID NO: 40 or988.

In some embodiments, the polynucleotide encodes a recombinant amylasecomprising a polypeptide sequence having at least 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NO: 156 or 1104, or a functional fragmentthereof, and wherein the encoded recombinant amylase comprises at leastone substitution or substitution set at one or more positions selectedfrom 37, 51, 91, 111, 112, 114, 122, 124, 128, 140, 167, 171, 183, 194,276, 280, 298, 397, and 464, wherein the amino acid positions of thepolypeptide sequence are numbered with reference to SEQ ID NO: 156 or1104. In some embodiments, the encoded recombinant amylase comprises atleast one substitution or substitution set selected from 37R, 51R, 91H,111A, 112Q, 114H, 122P, 122R, 124R, 128W, 140K, 167W, 171I, 183E, 183N,183V, 194E, 194R, 276A, 276K, 280L, 298K, 298M, 397T, and 464L, whereinthe amino acid positions of the polypeptide sequence are numbered withreference to SEQ ID NO: 156 or 1104. In some embodiments, the encodedrecombinant amylase comprises at least one substitution or substitutionset selected from N37R, G51R, K91H, E111A, K112Q, A114H, A122P, A122R,D124R, 1128W, R140K, E167W, E171I, G183E, G183N, G183V, L194E, L194R,T276A, T276K, Q280L, E298K, E298M, E397T, and N464L, wherein the aminoacid positions of the polypeptide sequence are numbered with referenceto SEQ ID NO: 156 or 1104.

In some embodiments, the polynucleotide encodes a recombinant amylasecomprising a polypeptide sequence having at least 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NO: 174 or 1122, or a functional fragmentthereof, and wherein the encoded recombinant amylase comprises at leastone substitution or substitution set at one or more positions selectedfrom 24, 91, 91/122, 91/122/128/130/167/171/409, 91/122/128/130/183/322,91/122/128/130/194, 91/122/128/130/322, 91/122/128/171/183/194/322,91/122/128/183/409, 91/122/130/167/183/194/409, 91/122/130/171/183,91/122/130/183/194, 91/122/130/194, 91/122/130/194/322,91/122/167/171/194, 91/122/167/171/194/322, 91/122/171/183/194/409,91/130/171/183/322/409, 91/167/171, 91/167/183/194, 91/167/322, 91/183,91/183/322/409, 91/194, 91/322, 91/409, 122/128, 122/128/194/409,122/130/171/322/409, 122/409, 137/145/227/394/480/481, 137/145/295,145/227/295/481, 145/309/481, 183, 227, 295/394, 322, 322/409, 394, and480/481, wherein the amino acid positions of the polypeptide sequenceare numbered with reference to SEQ ID NO: 174 or 1122. In someembodiments, the encoded recombinant amylase comprises at least onesubstitution or substitution set selected from 24V, 91H, 91H/122P,91H/122P/130D/183N/194R, 91H/122P/130D/194R/322A, 91H/122R,91H/122R/128W/130D/167W/171I/409S, 91H/122R/128W/130D/183N/322A,91H/122R/128W/130D/194R, 91H/122R/128W/130D/322A,91H/122R/128W/171I/183N/194R/322A, 91H/122R/128W/183N/409S,91H/122R/130D/167W/183N/194R/409S, 91H/122R/130D/171I/183N,91H/122R/130D/194R, 91H/122R/167W/171I/194R,91H/122R/167W/171I/194R/322A, 91H/122R/171I/183N/194R/409S,91H/130D/171I/183N/322A/409S, 91H/167W/171I, 91H/167W/183N/194R,91H/167W/322A, 91H/183N, 91H/183N/322A/409S, 91H/194R, 91H/322A,91H/409S, 122P/409S, 122R/128W, 122R/128W/194R/409S,122R/130D/171I/322A/409S, 137A/145G/227L/394E/480P/481V, 137A/145G/295N,145G/227L/295N/481V, 145G/309S/481V, 183N, 227L, 295N/394E, 322A,322A/409S, 394E, and 480T/481V, wherein the amino acid positions of thepolypeptide sequence are numbered with reference to SEQ ID NO: 174 or1122. In some embodiments, the encoded recombinant amylase comprises atleast one substitution or substitution set selected from L24V, K91H,K91H/A122P, K91H/A122P/E130D/G183N/L194R, K91H/A122P/E130D/L194R/F322A,K91H/A122R, K91H/A122R/I128W/E130D/E167W/E171I/V409S,K91H/A122R/I128W/E130D/G183N/F322A, K91H/A122R/I128W/E130D/L194R,K91H/A122R/I128W/E130D/F322A, K91H/A122R/I128W/E171I/G183N/L194R/F322A,K91H/A122R/I128W/G183N/V409S, K91H/A122R/E130D/E167W/G183N/L194R/V409S,K91H/A122R/E130D/E171I/G183N, K91H/A122R/E130D/L194R,K91H/A122R/E167W/E171I/L194R, K91H/A122R/E167W/E171I/L194R/F322A,K91H/A122R/E171I/G183N/L194R/V409S, K91H/E130D/E171I/G183N/F322A/V409S,K91H/E167W/E171I, K91H/E167W/G183N/L194R, K91H/E167W/F322A, K91H/G183N,K91H/G183N/F322A/V409S, K91H/L194R, K91H/F322A, K91H/V409S, A122P/V409S,A122R/I128W, A122R/I128W/L194R/V409S, A122R/E130D/E171I/F322A/V409S,G137A/A145G/T227L/Q394E/F480P/C481V, G137A/A145G/K295N,A145G/T227L/K295N/C481V, A145G/T309S/C481V, G183N, T227L, K295N/Q394E,F322A, F322A/V409S, Q394E, and F480T/C481V, wherein the amino acidpositions of the polypeptide sequence are numbered with reference to SEQID NO: 174 or 1122.

In some embodiments, the polynucleotide encodes a recombinant amylasecomprising a polypeptide sequence having at least 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NO: 276 or 1224, or a functional fragmentthereof, and wherein the encoded recombinant amylase comprises at leastone substitution or substitution set at one or more positions selectedfrom 16/26/29/128/145/256/309/383, 16/26/33/114/145/309/383/394,16/26/33/128/130/145/302/309/322/371, 16/26/33/145/158/383/394,16/26/145/302, 16/29/33/145/256/302, 18,26/29/33/130/145/157/158/183/394, 26/29/33/145/157/183/256/309/383,26/29/122/130/145/183/256/272/309/394, 26/29/145/157/256/302/322/481,26/29/145/256/272/302/309/322/481, 26/33/91/145/272/302/481,26/33/145/309/322/383, 26/114/145/322, 26/145/183/256/309/383,29/122/145/158/383, 29/130/145/256/272/371/394, 29/145/183/302/309/412,29/309/394, 33/122/145/272/309/412, 91/130/145/158/183/383, 91/309/383,130/145/256/272/371, 130/145/302/309, 130/272/481, 145/302/309, 268,309, 349, 360, 380, 394, 463, and 483, wherein the amino acid positionsof the polypeptide sequence are numbered with reference to SEQ ID NO:276 or 1224. In some embodiments, the encoded recombinant amylasecomprises at least one substitution or substitution set selected from16E/26E/29D/128I/145G/256A/309S/383Q,16E/26E/33D/114A/145G/309S/383Q/394E,16E/26E/33D/128I/130E/145G/302M/309S/322F/371Y,16E/26E/33D/145G/158K/383Q/394E, 16E/26E/145G/302M,16E/29D/33D/145G/256A/302M, 18S,26E/29D/33D/130E/145G/157F/158K/183G/394E,26E/29D/33D/145G/157F/183G/256A/309S/383Q,26E/29D/122A/130E/145G/183G/256A/272D/309S/394E,26E/29D/145G/157F/256A/302M/322F/481V,26E/29D/145G/256A/272D/302M/309S/322F/481V,26E/33D/91K/145G/272D/302M/481V, 26E/33D/145G/309S/322F/383Q,26E/114A/145G/322F, 26E/145G/183G/256A/309S/383Q,29D/122A/145G/158K/383Q, 29D/130E/145G/256A/272D/371Y/394E,29D/145G/183G/302M/309S/412L, 29D/309S/394E,33D/122A/145G/272D/309S/412L, 91K/130E/145G/158K/183G/383Q,91K/309S/383Q, 130E/145G/256A/272D/371Y, 130E/145G/302M/309S,130E/272D/481V, 145G/302M/309S, 268T, 309A, 349R, 360G, 380R, 394G,394N, 394S, 463M, and 483T, wherein the amino acid positions of thepolypeptide sequence are numbered with reference to SEQ ID NO: 276 or1224. In some embodiments, the encoded recombinant amylase comprises atleast one substitution or substitution set selected fromD16E/A26E/R29D/W128I/A145G/S256A/T309S/E383Q,D16E/A26E/E33D/H114A/A145G/T309S/E383Q/Q394E,D16E/A26E/E33D/W128I/D130E/A145G/Q302M/T309S/A322F/F371Y,D16E/A26E/E33D/A145G/R158K/E383Q/Q394E, D16E/A26E/A145G/Q302M,D16E/R29D/E33D/A145G/S256A/Q302M, G18S,A26E/R29D/E33D/D130E/A145G/Y157F/R158K/N183G/Q394E,A26E/R29D/E33D/A145G/Y157F/N183G/S256A/T309S/E383Q,A26E/R29D/R122A/D130E/A145G/N183G/S256A/G272D/T309S/Q394E,A26E/R29D/A145G/Y157F/S256A/Q302M/A322F/C481V,A26E/R29D/A145G/S256A/G272D/Q302M/T309S/A322F/C481V,A26E/E33D/H91K/A145G/G272D/Q302M/C481V,A26E/E33D/A145G/T309S/A322F/E383Q, A26E/H114A/A145G/A322F,A26E/A145G/N183G/S256A/T309S/E383Q, R29D/R122A/A145G/R158K/E383Q,R29D/D130E/A145G/S256A/G272D/F371Y/Q394E,R29D/A145G/N183G/Q302M/T309S/D412L, R29D/T309S/Q394E,E33D/R122A/A145G/G272D/T309S/D412L, H91K/D130E/A145G/R158K/N183G/E383Q,H91K/T309S/E383Q, D130E/A145G/S256A/G272D/F371Y,D130E/A145G/Q302M/T309S, D130E/G272D/C481V, A145G/Q302M/T309S, S268T,T309A, P349R, N360G, G380R, Q394G, Q394N, Q394S, G463M, and G483T,wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 276 or 1224.

In some embodiments, the polynucleotide encodes a recombinant amylasecomprising a polypeptide sequence having at least 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NO: 308 or 1256, or a functional fragmentthereof, and wherein the encoded recombinant amylase comprises at leastone substitution or substitution set at one or more positions selectedfrom 29, 37/124/250/270/349/380/394/442/491, 37/145,37/145/250/301/349/394, 37/145/258/442/491, 38/349/491, 81/259,81/313/495, 92/257/258/338/468, 92/258, 94/127/132/145/250/349,94/270/301/349/394/442, 94/349/491, 145/270, 178, 225, 226, 246,250/349/442, 259/313/338, 270/349, 286/338, 294, 313, 349, 349/380/442,413, 413/468, 448, and 473, wherein the amino acid positions of thepolypeptide sequence are numbered with reference to SEQ ID NO: 308 or1256. In some embodiments, the encoded recombinant amylase comprises atleast one substitution or substitution set selected from 29A,37T/124N/250D/270D/349R/380R/394E/442E/491L, 37T/145G,37T/145G/250D/301K/349R/394E, 37T/145G/258S/442E/491L, 38I/349R/491L,81E/259D, 81E/313D/495L, 92E/257E/258Q/338S/468T, 92E/258Q,94G/127E/132V/145G/250D/349R, 94G/270D/301K/349R/394E/442E,94G/349R/491L, 145G/270D, 178I, 225D, 226T, 246R, 250D/349R/442E,259D/313D/338S, 270D/349R, 286T/338S, 294L, 313D, 349R, 349R/380R/442E,413Y, 413Y/468I, 448T, and 473D, wherein the amino acid positions of thepolypeptide sequence are numbered with reference to SEQ ID NO: 308 or1256. In some embodiments, the encoded recombinant amylase comprises atleast one substitution or substitution set selected from R29A,N37T/D124N/E250D/E270D/P349R/G380R/G394E/D442E/I491L, N37T/A145G,N37T/A145G/E250D/R301K/P349R/G394E, N37T/A145G/K258S/D442E/I491L,M38I/P349R/I491L, A81E/P259D, A81E/T313D/I495L,R92E/P257E/K258Q/A338S/R468T, R92E/K258Q,D94G/Q127E/I132V/A145G/E250D/P349R, D94G/E270D/R301K/P349R/G394E/D442E,D94G/P349R/I491L, A145G/E270D, V178I, E225D, Q226T, K246R,E250D/P349R/D442E, P259D/T313D/A338S, E270D/P349R, M286T/A338S, M294L,T313D, P349R, P349R/G380R/D442E, F413Y, F413Y/R468I, H448T, and E473D,wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 308 or 1256.

In some embodiments, the polynucleotide encodes a recombinant amylasecomprising a polypeptide sequence having at least 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NO: 422 or 1370, or a functional fragmentthereof, and wherein the encoded recombinant amylase comprises at leastone substitution or substitution set at one or more positions selectedfrom 38, 38/178/226/286/338, 38/178/286/448, 38/178/349/448,38/226/246/258/286, 38/226/258/286/338/380/448, 38/226/258/349/413,38/226/286, 38/226/286/338/380, 38/226/338/349/380, 38/226/413/448,38/246/286, 38/246/286/380, 38/258, 38/286, 38/286/413/448, 38/286/448,38/448, 178, 178/226/258/286/310/338, 178/226/286/338, 178/246/338,178/258/286/413, 178/286, 178/286/338/349, 178/286/338/448, 178/380,178/413, 178/448, 184/246/258/380, 226, 226/246/286/349/448,226/246/413, 226/258/286/349/380, 226/258/286/413/448, 226/286,226/286/380/448, 226/349/380, 246, 246/286, 246/286/380/413, 246/338,246/413, 258/286/413, 286, 286/413/448, 338/448, 349, and 413, whereinthe amino acid positions of the polypeptide sequence are numbered withreference to SEQ ID NO: 422 or 1370. In some embodiments, the encodedrecombinant amylase comprises at least one substitution or substitutionset selected from 38I, 38I/178I/226T/286T/338S, 38I/178I/286T/448T,38I/178I/349R/448T, 38I/226T/246R/258S/286T,38I/226T/258S/286T/338S/380R/448T, 38I/226T/258S/349R/413Y,38I/226T/286T, 38I/226T/286T/338S/380R, 38I/226T/338S/349R/380R,38I/226T/413Y/448T, 38I/246R/286T, 38I/246R/286T/380R, 38I/258S,38I/286T, 38I/286T/413Y/448T, 38I/286T/448T, 38I/448T, 178I,178I/226T/258S/286T/310R/338S, 178I/226T/286T/338S, 178I/246R/338S,178I/258S/286T/413Y, 178I/286T, 178I/286T/338S/349R,178I/286T/338S/448T, 178I/380R, 178I/413Y, 178I/448T,184K/246R/258S/380R, 226T, 226T/246R/286T/349R/448T, 226T/246R/413Y,226T/258S/286T/349R/380R, 226T/258S/286T/413Y/448T, 226T/286T,226T/286T/380R/448T, 226T/349R/380R, 246R, 246R/286T,246R/286T/380R/413Y, 246R/338S, 246R/413Y, 246T, 258S/286T/413Y, 286T,286T/413Y/448T, 338S/448T, 349R, and 413Y, wherein the amino acidpositions of the polypeptide sequence are numbered with reference to SEQID NO: 422 or 1370. In some embodiments, the encoded recombinant amylasecomprises at least one substitution or substitution set selected fromM38I, M38I/V178I/Q226T/M286T/A338S, M38I/V178I/M286T/H448T,M38I/V178I/P349R/H448T, M38I/Q226T/K246R/K258S/M286T,M38I/Q226T/K258S/M286T/A338S/G380R/H448T, M38I/Q226T/K258S/P349R/F413Y,M38I/Q226T/M286T, M38I/Q226T/M286T/A338S/G380R,M38I/Q226T/A338S/P349R/G380R, M38I/Q226T/F413Y/H448T, M38I/K246R/M286T,M38I/K246R/M286T/G380R, M38I/K258S, M38I/M286T, M38I/M286T/F413Y/H448T,M38I/M286T/H448T, M38/1H448T, V178I,V178I/Q226T/K258S/M286T/Q310R/A338S, V178I/Q226T/M286T/A338S,V178I/K246R/A338S, V178I/K258S/M286T/F413Y, V178I/M286T,V178I/M286T/A338S/P349R, V178I/M286T/A338S/H448T, V178I/G380R,V178I/F413Y, V178I/H448T, E184K/K246R/K258S/G380R, Q226T,Q226T/K246R/M286T/P349R/H448T, Q226T/K246R/F413Y,Q226T/K258S/M286T/P349R/G380R, Q226T/K258S/M286T/F413Y/H448T,Q226T/M286T, Q226T/M286T/G380R/H448T, Q226T/P349R/G380R, K246R,K246R/M286T, K246R/M286T/G380R/F413Y, K246R/A338S, K246R/F413Y, K246T,K258S/M286T/F413Y, M286T, M286T/F413Y/H448T, A338S/H448T, P349R, andF413Y, wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 422 or 1370.

In some embodiments, the polynucleotide encodes a recombinant amylasecomprising a polypeptide sequence having at least 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NO: 456 or 1404, or a functional fragmentthereof, and wherein the encoded recombinant amylase comprises at leastone substitution or substitution set at one or more positions selectedfrom 29/31/92/220, 29/469, 49/58/103/141/197/269/294/298,49/58/103/194/204/298, 49/58/141/298, 49/103, 103, 123, 126, 127, 131,134, 180, 210, 214, 220/469, 228, 259, 279, 282, 302, 319, 393, 415,417, 428, 429, 466, and 469, wherein the amino acid positions of thepolypeptide sequence are numbered with reference to SEQ ID NO: 456 or1404. In some embodiments, the encoded recombinant amylase comprises atleast one substitution or substitution set selected from29E/31L/92A/220G, 29E/469I, 49A/58T/103L/141F/197Y/269F/294H/298Q,49A/58T/103L/194F/204D/298H, 49A/58T/141F/298H, 49A/103L, 103L, 123R,126R, 127W, 131H, 131R, 131Y, 134R, 180E, 210P, 214G, 220G/469I, 228V,259C, 279L, 282T, 302H, 302R, 302W, 319S, 393R, 393S, 415C, 417W, 428T,429S, 466C, and 469I, wherein the amino acid positions of thepolypeptide sequence are numbered with reference to SEQ ID NO: 456 or1404. In some embodiments, the encoded recombinant amylase comprises atleast one substitution or substitution set selected fromR29E/F31L/R92A/A220G, R29E/V469I,S49A/S58T/V103L/Y141F/F197Y/H269F/M294H/E298Q,S49A/S58T/V103L/L194F/N204D/E298H, S49A/S58T/Y141F/E298H, S49A/V103L,V103L, D123R, T126R, Q127W, E131H, E131R, E131Y, E134R, D180E, H210P,E214G, A220G/V469I, Q228V, P259C, D279L, E282T, Q302H, Q302R, Q302W,A319S, E393R, E393S, D415C, P417W, D428T, E429S, Q466C, and V469I,wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 456 or 1404.

In some embodiments, the polynucleotide encodes a recombinant amylasecomprising a polypeptide sequence having at least 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NO: 456 or 1404, or a functional fragmentthereof, and wherein the encoded recombinant amylase comprises at leastone substitution or substitution set at one or more positions selectedfrom 92, 123, 126, 127, 129, 131, 134, 140, 172, 180, 210, 214, 221,228, 253, 254, 259, 279, 282, 302, 304, 305, 319, 322, 346, 359, 384,385, 387, 393, 409, 412, 415, 417, 428, 429, 441, 442, 444, 446, 451,455, 459, 466, 467, 470, 473, 474, 492, and 494, wherein the amino acidpositions of the polypeptide sequence are numbered with reference to SEQID NO: 456 or 1404. In some embodiments, the encoded recombinant amylasecomprises at least one substitution or substitution set selected from92G, 92Q, 123C, 123G, 123R, 126Q, 126R, 1271, 127R, 127S, 127V, 127W,129C, 129W, 131H, 131R, 131Y, 134A, 134F, 134R, 140V, 172K, 172R, 180E,180P, 180Q, 210G, 210P, 214G, 214L, 214R, 221T, 221V, 228V, 253R, 254G,259C, 259R, 259W, 279L, 279V, 282R, 282T, 302G, 302H, 302R, 302V, 302W,304T, 305Q, 305R, 305W, 319L, 319S, 322R, 346G, 359G, 384M, 385L, 387G,387V, 393R, 393S, 393W, 409A, 409R, 409W, 412A, 415C, 415G, 417W, 428T,429S, 441R, 441W, 442R, 442W, 444G, 444R, 444V, 446G, 451F, 455L, 455R,455T, 455W, 459W, 466C, 467P, 467W, 470G, 470L, 470S, 473M, 474G, 492G,492R, and 494C, wherein the amino acid positions of the polypeptidesequence are numbered with reference to SEQ ID NO: 456. In someembodiments, the encoded recombinant amylase comprises at least onesubstitution or substitution set selected from R92G, R92Q, D123C, D123G,D123R, T126Q, T126R, Q1271, Q127R, Q127S, Q127V, Q127W, D129C, D129W,E131H, E131R, E131Y, E134A, E134F, E134R, R140V, D172K, D172R, D180E,D180P, D180Q, H210G, H210P, E214G, E214L, E214R, R221T, R221V, Q228V,Q253R, E254G, P259C, P259R, P259W, D279L, D279V, E282R, E282T, Q302G,Q302H, Q302R, Q302V, Q302W, R304T, D305Q, D305R, D305W, A319L, A319S,A322R, W346G, E359G, T384M, Y385L, 1387G, 1387V, E393R, E393S, E393W,V409A, V409R, V409W, D412A, D415C, D415G, P417W, D428T, E429S, D441R,D441W, D442R, D442W, E444G, E444R, E444V, T446G, E451F, N455L, N455R,N455T, N455W, R459W, Q466C, E467P, E467W, V470G, V470L, V470S, E473M,N474G, E492G, E492R, and V494C, wherein the amino acid positions of thepolypeptide sequence are numbered with reference to SEQ ID NO: 456 or1404.

In some embodiments, the polynucleotide encodes a recombinant amylasecomprising a polypeptide sequence having at least 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NO: 546 or 1494, or a functional fragmentthereof, and wherein the encoded recombinant amylase comprises at leastone substitution or substitution set at one or more positions selectedfrom 123, 123/126/180/214/298/302/393, 123/126/180/214/393/428,123/126/210/298/302/393, 123/126/210/428, 123/126/428, 123/210/302,126/180/210/214/298/302, 126/180/210/214/428, 126/180/298/302/393,126/210/302/428, 126/214/393, 126/298/302/393, 126/298/302/393/428, 129,129/228/253, 131/228/279, 131/282, 180/214, 210/214/302/393/428,228/282/444, 253/279/470, 253/279/492, 279, 279/470, and 441, whereinthe amino acid positions of the polypeptide sequence are numbered withreference to SEQ ID NO: 546 or 1494. In some embodiments, the encodedrecombinant amylase comprises at least one substitution or substitutionset selected from 123R, 123R/126R/180E/214L/298Q/302W/393R,123R/126R/180Q/214G/393R/428T, 123R/126R/210P/298Q/302W/393R,123R/126R/210P/428T, 123R/126R/428T, 123R/210P/302R,126R/180E/210P/214G/428T, 126R/180E/298Q/302R/393R,126R/180Q/210P/214L/298Q/302R, 126R/210P/302R/428T, 126R/214G/393R,126R/298Q/302H/393R, 126R/298Q/302R/393R/428T, 129W, 129W/228V/253R,131H/228V/279V, 131Y/282T, 180Q/214L, 210P/214L/302R/393R/428T,228V/282T/444R, 253R/279L/492G, 253R/279V/470S, 279L, 279L/470S, and441R, wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 546 or 1494. In some embodiments,the encoded recombinant amylase comprises at least one substitution orsubstitution set selected from D123R,D123R/T126R/D180E/E214L/H298Q/Q302W/E393R,D123R/T126R/D180Q/E214G/E393R/D428T,D123R/T126R/H210P/H298Q/Q302W/E393R, D123R/T126R/H210P/D428T,D123R/T126R/D428T, D123R/H210P/Q302R, T126R/D180E/H210P/E214G/D428T,T126R/D180E/H298Q/Q302R/E393R, T126R/D180Q/H210P/E214L/H298Q/Q302R,T126R/H210P/Q302R/D428T, T126R/E214G/E393R, T126R/H298Q/Q302H/E393R,T126R/H298Q/Q302R/E393R/D428T, D129W, D129W/Q228V/Q253R,E131H/Q228V/D279V, E131Y/E282T, D180Q/E214L,H210P/E214L/Q302R/E393R/D428T, Q228V/E282T/E444R, Q253R/D279L/E492G,Q253R/D279V/V470S, D279L, D279L/V470S, and D441R, wherein the amino acidpositions of the polypeptide sequence are numbered with reference to SEQID NO: 546 or 1494.

In some embodiments, the polynucleotide encodes a recombinant amylasecomprising a polypeptide sequence having at least 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NO: 632 or 1580, or a functional fragmentthereof, and wherein the encoded recombinant amylase comprises at leastone substitution or substitution set at one or more positions selectedfrom 2, 45/166/298, 48, 48/166/170/203, 52, 58/166, 58/203, 72, 82, 114,114/223/306/308, 149, 165, 166/203, 167, 170, 184/216/306/308,184/223/306/437, 189, 203, 225, 255, 258, 275, 280, 284, 294, 298, 396,397, 411, 431, 448, 468, 482, 483, and 495, wherein the amino acidpositions of the polypeptide sequence are numbered with reference to SEQID NO: 632. In some embodiments, the encoded recombinant amylasecomprises at least one substitution or substitution set selected from2L, 45S/166L/298D, 48D, 48D/166L/170S/203S, 52R, 58A/166L, 58A/203S,72T, 82A, 114R, 114R/223L/306F/308L, 149K, 165V, 166L/203S, 167Y, 170S,184D/216L/306F/308L, 184D/223L/306F/437L, 189D, 203S, 225T, 255M, 255R,258S, 275I, 275R, 280C, 284L, 284M, 294A, 298D, 298N, 396E, 396Q, 397M,411E, 431T, 448P, 468V, 482L, 483A, 495G, and 495L, wherein the aminoacid positions of the polypeptide sequence are numbered with referenceto SEQ ID NO: 632 or 1580. In some embodiments, the encoded recombinantamylase comprises at least one substitution or substitution set selectedfrom R2L, Y45S/I166L/H298D, A48D, A48D/I166L/D170S/E203S, Y52R,T58A/I166L, T58A/E203S, S72T, Q82A, H114R, H114R/V223L/Y306F/M308L,Q149K, H165V, I166L/E203S, E167Y, D170S, E184D/I216L/Y306F/M308L,E184D/V223L/Y306F/M437L, Q189D, E203S, E225T, V255M, V255R, K258S,Q275I, Q275R, Q280C, K284L, K284M, M294A, H298D, H298N, D396E, D396Q,E397M, T411E, P431T, H448P, R468V, N482L, G483A, I495G, and I495L,wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 632 or 1580.

In some embodiments, the polynucleotide encodes a recombinant amylasecomprising a polypeptide sequence having at least 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NO: 664 or 1612, or a functional fragmentthereof, and wherein the encoded recombinant amylase comprises at leastone substitution or substitution set at one or more positions selectedfrom 6/39/103/324/420, 41, 58/500/501, 61, 95/98/205/227/423/458,95/98/227/423, 95/205, 95/252/285/324, 123/170/197/275/298/396/448/483,123/197, 123/197/225/294/396/448/469, 123/284/294/324/396/448,134/225/275/302, 165/225/284/298/469/483, 170/275/294/298/448, 205/206,205/206/224/315/458/480, 206/227, 206/227/315/423, 225/294/298/396, 227,392/503, 458, 499, and 500, wherein the amino acid positions of thepolypeptide sequence are numbered with reference to SEQ ID NO: 664 or1612. In some embodiments, the encoded recombinant amylase comprises atleast one substitution or substitution set selected from6I/39I/103D/324T/420V, 41F, 58S/500P/501P, 61Y, 95L/252I/285V/324T,95V/98I/205V/227V/423V/458F, 95V/98I/227V/423V, 95V/205V,123E/170S/197Y/275I/298N/396Q/448Q/483Q, 123E/197Y,123E/197Y/225G/294A/396Q/448Q/469I, 123E/284L/294A/324T/396Q/448Q,134R/225G/275I/302W, 165V/225G/284L/298N/469I/483Q,170S/275I/294A/298N/448Q, 205V/206E, 205V/206E/224L/315S/458F/480L,206E/227V, 206E/227V/315S/423V, 225G/294A/298N/396Q, 227C, 392C/503L,458F, 458Y, 499R, and 500P, wherein the amino acid positions of thepolypeptide sequence are numbered with reference to SEQ ID NO: 664 or1612. In some embodiments, the encoded recombinant amylase comprises atleast one substitution or substitution set selected fromL6I/V39I/V103D/A324T/I420V, L41F, T58S/H500P/H501P, L61Y,I95L/V252I/T285V/A324T, I95V/L98I/I205V/T227V/S423V/W458F,I95V/L98I/T227V/S423V, I95V/I205V,D123E/D170S/F197Y/Q275I/H298N/D396Q/H448Q/G483Q, D123E/F197Y,D123E/F197Y/E225G/M294A/D396Q/H448Q/V469I,D123E/K284L/M294A/A324T/D396Q/H448Q, E134R/E225G/Q275I/Q302W,H165V/E225G/K284L/H298N/V469I/G483Q, D170S/Q275I/M294A/H298N/H448Q,I205V/D206E, I205V/D206E/M224L/T315S/W458F/F480L, D206E/T227V,D206E/T227V/T315S/S423V, E225G/M294A/H298N/D396Q, T227C, I392C/H503L,W458F, W458Y, G499R, and H500P, wherein the amino acid positions of thepolypeptide sequence are numbered with reference to SEQ ID NO: 664 or1612.

In some embodiments, the polynucleotide encodes a recombinant amylasecomprising a polypeptide sequence having at least 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NO: 664 or 1612, or a functional fragmentthereof, and wherein the encoded recombinant amylase comprises at leastone substitution or substitution set at one or more positions selectedfrom 95/252/285/324, 123/197, 123/197/225/294/396/448/469,134/225/275/302, 205/206, and 205/206/224/315/458/480, wherein the aminoacid positions of the polypeptide sequence are numbered with referenceto SEQ ID NO: 664 or 1612. In some embodiments, the encoded recombinantamylase comprises at least one substitution or substitution set selectedfrom 95L/252I/285V/324T, 123E/197Y, 123E/197Y/225G/294A/396Q/448Q/469I,134R/225G/275I/302W, 205V/206E, and 205V/206E/224L/315S/458F/480L,wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 664 or 1612. In some embodiments,the encoded recombinant amylase comprises at least one substitution orsubstitution set selected from I95L/V252I/T285V/A324T, D123E/F197Y,D123E/F197Y/E225G/M294A/D396Q/H448Q/V469I, E134R/E225G/Q275I/Q302W,I205V/D206E, and I205V/D206E/M224L/T315S/W458F/F480L, wherein the aminoacid positions of the polypeptide sequence are numbered with referenceto SEQ ID NO: 664 or 1612.

In some embodiments, the polynucleotide encodes a recombinant amylasecomprising a polypeptide sequence having at least 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NO: 780 or 1728, or a functional fragmentthereof, and wherein the encoded recombinant amylase comprises asubstitution at position 126, wherein the amino acid positions of thepolypeptide sequence are numbered with reference to SEQ ID NO: 780 or1728. In some embodiments, the encoded recombinant amylase comprises atleast one substitution selected from 126C, 126D, 126E, 126K, 126L, 126M,126N, 126Q, 126S, 126T, and 126W, wherein the amino acid positions ofthe polypeptide sequence are numbered with reference to SEQ ID NO: 780or 1728. In some embodiments, the encoded recombinant amylase comprisesat least one substitution selected from R126C, R126D, R126E, R126K,R126L, R126M, R126N, R126Q, R126S, R126T, and R126W, wherein the aminoacid positions of the polypeptide sequence are numbered with referenceto SEQ ID NO: 780 or 1728.

In some embodiments, the polynucleotides are capable of hybridizingunder highly stringent conditions to a reference polynucleotidesequence. In some embodiments, the reference sequence is selected fromSEQ ID NOS: 1, 17, 39, 155, 173, 275, 307, 421, 455, 545, 631, 663, 779,987, 1103, 1121, 1223, 1255, 1369, 1403, 1493, 1579, 1611, and 1727, ora complement thereof, or a polynucleotide sequence encoding any of thevariant amylase polypeptides provided herein. In some embodiments, thepolynucleotide capable of hybridizing under highly stringent conditionsencodes a amylase polypeptide comprising an amino acid sequence that hasone or more residue differences as compared to SEQ ID NO: 1, 17, 39,155, 173, 275, 307, 421, 455, 545, 631, 663, 779, 987, 1103, 1121, 1223,1255, 1369, 1403, 1493, 1579, 1611, and 1727 at residue positionsselected from any positions as set forth in Table 4-1, 4-2, 4-3, 4-4,4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 4-11, 4-12, 4-13, and/or 4-14. In somefurther embodiments, the engineered polynucleotide is selected fromthose provided in Table 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7, 4-8, 4-9,4-10, 4-11, 4-12, 4-13, and/or 4-14, or comprises a polynucleotidehaving at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity to a reference sequence selected from SEQ ID NO: 1, 17, 39,155, 173, 275, 307, 421, 455, 545, 631, 663, 779, 987, 1103, 1121, 1223,1255, 1369, 1403, 1493, 1579, 1611, and 1727. In some additionalembodiments, the engineered polynucleotide comprises a sequence havingat least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity to at least one polynucleotide sequence provided in Table 4-1,4-2, 4-3, 4-4, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 4-11, 4-12, 4-13, and/or4-14, or at least one polynucleotide sequence provided in Table 4-1,4-2, 4-3, 4-4, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 4-11, 4-12, 4-13, and/or4-14, wherein the polynucleotide sequence lacks the sequence encodingthe histidine tag and the preceding amino acid linker, and/or SEQ ID NO:1, 17, 39, 155, 173, 275, 307, 421, 455, 545, 631, 663, 779, 987, 1103,1121, 1223, 1255, 1369, 1403, 1493, 1579, 1611, and 1727. In somefurther embodiments, the engineered polynucleotide sequence comprises1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-21, 1-22,1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45, or 1-50 base changes, ascompared to a reference polynucleotide sequence. In still some otherembodiments, the engineered polynucleotide sequence comprises 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 30, 31, 32, 33, 34, 35, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, or 50 base changes, as compared to a reference polynucleotidesequence. In some embodiments, the engineered polynucleotide sequencecomprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20,21, 22, 23, 24, or 25 base changes, as compared to a referencepolynucleotide sequence.

In some embodiments, an isolated polynucleotide encoding any of theengineered amylase polypeptides provided herein is manipulated in avariety of ways to provide for expression of the gene and production ofthe polypeptide. In some embodiments, the polynucleotides encoding thepolypeptides are provided as expression vectors, in which one or morecontrol sequences is present to regulate the expression of thepolynucleotides and/or polypeptides. Manipulation of the isolatedpolynucleotide prior to its insertion into a vector may be desirable ornecessary depending on the expression vector. The techniques formodifying polynucleotides and nucleic acid sequences utilizingrecombinant DNA methods are well known in the art.

In some embodiments, the control sequences include among othersequences, promoters, leader sequences, polyadenylation sequences,propeptide sequences, signal peptide sequences, and transcriptionterminators. As known in the art, suitable control sequences can beselected based on the host cells used. It is not intended that thepresent invention be limited to any specific control sequences.

Exemplary promoters for bacterial host cells include, but are notlimited to promoters obtained from the genes for Escherichia colilactose operon, tryptophan operon, arabinose operon, T7 promoter fromthe T7 bacteriophage, and Saccharopolyspora erythraea erythromycinresistance gene.

Exemplary promoters for filamentous fungal host cells, include promotersobtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucormiehei aspartic proteinase, Aspergillus niger neutral alpha-amylase,Aspergillus niger acid stable alpha-amylase, Aspergillus niger orAspergillus awamori glucoamylase, Rhizomucor miehei protease,Aspergillus oryzae alkaline protease, Aspergillus oryzae triosephosphate isomerase, Aspergillus nidulans acetamidase, and Fusariumoxysporum trypsin-like protease (See e.g., WO 96/00787), as well as theNA2-tpi promoter (a hybrid of the promoters from the genes forAspergillus niger neutral alpha-amylase and Aspergillus oryzae triosephosphate isomerase), and mutant, truncated, and hybrid promotersthereof. Exemplary yeast cell promoters can be from the genes can befrom the genes for Saccharomyces cerevisiae enolase (ENO-1),Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiaealcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase(ADH2/GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase.Other useful promoters for yeast host cells are known in the art (Seee.g., Romanos et al., Yeast 8:423-488 [1992]). Exemplary promoters foruse in mammalian cells include, but are not limited to those fromcytomegalovirus (CMV), Simian vacuolating virus 40 (SV40), from Homosapiens phosphoglycerate kinase, beta actin, elongation factor-1a orglyceraldehyde-3-phosphate dehydrogenase, or from Gallus gallus'β-actin.

In some embodiments, the control sequence is a suitable transcriptionterminator sequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleic acid sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice finds use inthe present invention. For example, exemplary transcription terminatorsfor bacterial host cells can be obtained from the T7 bacterial phage forthe T7 terminator, or Escherichia coli ribosomal RNA, for example therrnB terminator. For example, exemplary transcription terminators forfilamentous fungal host cells can be obtained from the genes forAspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase,Aspergillus nidulans anthranilate synthase, Aspergillus nigeralpha-glucosidase, and Fusarium oxysporum trypsin-like protease.Exemplary terminators for yeast host cells can be obtained from thegenes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are known in the art (See e.g., Romanos et al., supra).Exemplary terminators for mammalian cells include, but are not limitedto those from cytomegalovirus (CMV), Simian vacuolating virus 40 (SV40),or from Homo sapiens growth hormone.

In some embodiments, the control sequence is a suitable leader sequence,a non-translated region of an mRNA that is important for translation bythe host cell. The leader sequence is operably linked to the 5′ terminusof the nucleic acid sequence encoding the polypeptide. Any leadersequence that is functional in the host cell of choice may be used.Exemplary leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase. Suitable leaders for yeast host cellsinclude, but are not limited to those obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

In some embodiments, the control sequence may also be a polyadenylationsequence, a sequence operably linked to the 3′ terminus of the nucleicacid sequence and which, when transcribed, is recognized by the hostcell as a signal to add polyadenosine residues to transcribed mRNA. Anypolyadenylation sequence which is functional in the host cell of choicemay be used in the present invention. Exemplary polyadenylationsequences for filamentous fungal host cells include, but are not limitedto those from the genes for Aspergillus oryzae TAKA amylase, Aspergillusniger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusariumoxysporum trypsin-like protease, and Aspergillus nigeralpha-glucosidase. Useful polyadenylation sequences for yeast host cellsare also known in the art (See e.g., Guo and Sherman, Mol. Cell. Biol.,15:5983-5990 [1995]).

In some embodiments, the control sequence is a signal peptide codingregion that codes for an amino acid sequence linked to the aminoterminus of a polypeptide and directs the encoded polypeptide into thecell's secretory pathway. The 5′ end of the coding sequence of thenucleic acid sequence may inherently contain a signal peptide codingregion naturally linked in translation reading frame with the segment ofthe coding region that encodes the secreted polypeptide. Alternatively,the 5′ end of the coding sequence may contain a signal peptide codingregion that is foreign to the coding sequence.

Any signal peptide coding region that directs the expressed polypeptideinto the secretory pathway of a host cell of choice finds use forexpression of the engineered amylase polypeptides provided herein.Effective signal peptide coding regions for filamentous fungal hostcells include, but are not limited to the signal peptide coding regionsobtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillusniger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor mieheiaspartic proteinase, Humicola insolens cellulase, and Humicolalanuginosa protease. Useful signal peptides for yeast host cellsinclude, but are not limited to those from the genes for Saccharomycescerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Usefulsignal peptides for mammalian host cells include but are not limited tothose from the genes for immunoglobulin gamma (IgG).

In some embodiments, the control sequence is a propeptide coding regionthat codes for an amino acid sequence positioned at the amino terminusof a polypeptide. The resultant polypeptide is referred to as a“proenzyme,” “propolypeptide,” or “zymogen,” in some cases). Apropolypeptide can be converted to a mature active polypeptide bycatalytic or autocatalytic cleavage of the propeptide from thepropolypeptide.

In another aspect, the present invention also provides a recombinantexpression vector comprising a polynucleotide encoding an engineeredamylase polypeptide, and one or more expression regulating regions suchas a promoter and a terminator, a replication origin, etc., depending onthe type of hosts into which they are to be introduced. in someembodiments, the various nucleic acid and control sequences describedabove are joined together to produce a recombinant expression vectorwhich includes one or more convenient restriction sites to allow forinsertion or substitution of the nucleic acid sequence encoding thevariant amylase polypeptide at such sites. Alternatively, thepolynucleotide sequence(s) of the present invention are expressed byinserting the polynucleotide sequence or a nucleic acid constructcomprising the polynucleotide sequence into an appropriate vector forexpression. In creating the expression vector, the coding sequence islocated in the vector so that the coding sequence is operably linkedwith the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus), that can be conveniently subjected to recombinant DNA proceduresand can result in the expression of the variant amylase polynucleotidesequence. The choice of the vector will typically depend on thecompatibility of the vector with the host cell into which the vector isto be introduced. The vectors may be linear or closed circular plasmids.

In some embodiments, the expression vector is an autonomouslyreplicating vector (i.e., a vector that exists as an extra-chromosomalentity, the replication of which is independent of chromosomalreplication, such as a plasmid, an extra-chromosomal element, aminichromosome, or an artificial chromosome). The vector may contain anymeans for assuring self-replication. In some alternative embodiments,the vector may be one which, when introduced into the host cell, isintegrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids which togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon may be used.

In some embodiments, the expression vector preferably contains one ormore selectable markers, which permit easy selection of transformedcells.

A “selectable marker” is a gene the product of which provides forbiocide or viral resistance, resistance to heavy metals, prototrophy toauxotrophs, and the like. Suitable markers for bacterial host cellsinclude, but are not limited to carbenicillin, ampicillin,chloramphenicol, tetracyclineJD, kanamycin and zeocin.

Suitable markers for yeast host cells include, but are not limited toADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for usein a filamentous fungal host cell include, but are not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferases), bar(phosphinothricin acetyltransferase), hph (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof. Inanother aspect, the present invention provides a host cell comprising apolynucleotide encoding at least one engineered amylase polypeptide ofthe present application, the polynucleotide being operatively linked toone or more control sequences for expression of the engineered amylaseenzyme(s) in the host cell.

Host cells for use in expressing the polypeptides encoded by theexpression vectors of the present invention are well known in the artand include but are not limited to, bacterial cells (e.g., E. coli);fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae andPichia pastoris [e.g., ATCC Accession No. 201178]); insect cells (e.g.,Drosophila S2 and Spodoptera Sf9 cells), plant cells, animal cells(e.g., CHO, COS, and BHK), and human cells (e.g., HEK293T, humanfibroblast, THP-1, Jurkat and Bowes melanoma cell lines).

Accordingly, in another aspect, the present invention provides methodsfor producing the engineered amylase polypeptides, where the methodscomprise culturing a host cell capable of expressing a polynucleotideencoding the engineered amylase polypeptide under conditions suitablefor expression of the polypeptide. In some embodiments, the methodsfurther comprise the steps of isolating and/or purifying the amylasepolypeptides, as described herein.

Appropriate culture media and growth conditions for the above-describedhost cells are well known in the art. Polynucleotides for expression ofthe amylase may be introduced into cells by various methods known in theart. Techniques include, among others, electroporation, biolisticparticle bombardment, liposome mediated transfection, calcium chloridetransfection, and protoplast fusion.

The engineered amylase with the properties disclosed herein can beobtained by subjecting the polynucleotide encoding the naturallyoccurring or engineered amylase polypeptide to mutagenesis and/ordirected evolution methods known in the art, and as described herein. Anexemplary directed evolution technique is mutagenesis and/or DNAshuffling (See e.g., Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-10751[1994]; WO 95/22625; WO 97/0078; WO 97/35966; WO 98/27230; WO 00/42651;WO 01/75767 and U.S. Pat. No. 6,537,746). Other directed evolutionprocedures that can be used include, among others, staggered extensionprocess (StEP), in vitro recombination (See e.g., Zhao et al., Nat.Biotechnol., 16:258-261 [1998]), mutagenic PCR (See e.g., Caldwell etal., PCR Methods Appl., 3:S136-S140 [1994]), and cassette mutagenesis(See e.g., Black et al., Proc. Natl. Acad. Sci. USA 93:3525-3529[1996]).

Recombinant polypeptides can be produced using any suitable methodsknown in the art. Genes encoding the wild-type polypeptide of interestcan be cloned in vectors, such as plasmids, and expressed in desiredhosts, such as E. coli, etc. Variants of recombinant polypeptides can begenerated by various methods known in the art. Indeed, there is a widevariety of different mutagenesis techniques well known to those skilledin the art. In addition, mutagenesis kits are also available from manycommercial molecular biology suppliers. Methods are available to makespecific substitutions at defined amino acids (site-directed), specificor random mutations in a localized region of the gene (regio-specific),or random mutagenesis over the entire gene (e.g., saturationmutagenesis). Numerous suitable methods are known to those in the art togenerate enzyme variants, including but not limited to site-directedmutagenesis of single-stranded DNA or double-stranded DNA using PCR,cassette mutagenesis, gene synthesis, error-prone PCR, shuffling, andchemical saturation mutagenesis, or any other suitable method known inthe art. Mutagenesis and directed evolution methods can be readilyapplied to enzyme-encoding polynucleotides to generate variant librariesthat can be expressed, screened, and assayed. Any suitable mutagenesisand directed evolution methods find use in the present invention and arewell known in the art (See e.g., U.S. Pat. Nos. 5,605,793, 5,811,238,5,830,721, 5,834,252, 5,837,458, 5,928,905, 6,096,548, 6,117,679,6,132,970, 6,165,793, 6,180,406, 6,251,674, 6,265,201, 6,277,638,6,287,861, 6,287,862, 6,291,242, 6,297,053, 6,303,344, 6,309,883,6,319,713, 6,319,714, 6,323,030, 6,326,204, 6,335,160, 6,335,198,6,344,356, 6,352,859, 6,355,484, 6,358,740, 6,358,742, 6,365,377,6,365,408, 6,368,861, 6,372,497, 6,337,186, 6,376,246, 6,379,964,6,387,702, 6,391,552, 6,391,640, 6,395,547, 6,406,855, 6,406,910,6,413,745, 6,413,774, 6,420,175, 6,423,542, 6,426,224, 6,436,675,6,444,468, 6,455,253, 6,479,652, 6,482,647, 6,483,011, 6,484,105,6,489,146, 6,500,617, 6,500,639, 6,506,602, 6,506,603, 6,518,065,6,519,065, 6,521,453, 6,528,311, 6,537,746, 6,573,098, 6,576,467,6,579,678, 6,586,182, 6,602,986, 6,605,430, 6,613,514, 6,653,072,6,686,515, 6,703,240, 6,716,631, 6,825,001, 6,902,922, 6,917,882,6,946,296, 6,961,664, 6,995,017, 7,024,312, 7,058,515, 7,105,297,7,148,054, 7,220,566, 7,288,375, 7,384,387, 7,421,347, 7,430,477,7,462,469, 7,534,564, 7,620,500, 7,620,502, 7,629,170, 7,702,464,7,747,391, 7,747,393, 7,751,986, 7,776,598, 7,783,428, 7,795,030,7,853,410, 7,868,138, 7,783,428, 7,873,477, 7,873,499, 7,904,249,7,957,912, 7,981,614, 8,014,961, 8,029,988, 8,048,674, 8,058,001,8,076,138, 8,108,150, 8,170,806, 8,224,580, 8,377,681, 8,383,346,8,457,903, 8,504,498, 8,589,085, 8,762,066, 8,768,871, 8,849,575,9,593,326, 9,665,694, 9,684,771, 9,864,833, 9,996,661, and all relatedUS, as well as PCT and non-US counterparts; Ling et al., Anal. Biochem.,254(2):157-78 [1997]; Dale et al., Meth. Mol. Biol., 57:369-74 [1996];Smith, Ann. Rev. Genet., 19:423-462 [1985]; Botstein et al., Science,229:1193-1201 [1985]; Carter, Biochem. J., 237:1-7 [1986]; Kramer etal., Cell, 38:879-887 [1984]; Wells et al., Gene, 34:315-323 [1985];Minshull et al., Curr. Op. Chem. Biol., 3:284-290 [1999]; Christians etal., Nat. Biotechnol., 17:259-264 [1999]; Crameri et al., Nature,391:288-291 [1998]; Crameri, et al., Nat. Biotechnol., 15:436-438[1997]; Zhang et al., Proc. Nat. Acad. Sci. U.S.A., 94:4504-4509 [1997];Crameri et al., Nat. Biotechnol., 14:315-319 [1996]; Stemmer, Nature,370:389-391 [1994]; Stemmer, Proc. Nat. Acad. Sci. USA, 91:10747-10751[1994]; WO 95/22625; WO 97/0078; WO 97/35966; WO 98/27230; WO 00/42651;WO 01/75767; and WO 2009/152336, all of which are incorporated herein byreference).

In some embodiments, the enzyme variants obtained following mutagenesistreatment are screened by subjecting the enzyme variants to a definedtemperature (or other assay conditions) and measuring the amount ofenzyme activity remaining after heat treatments or other assayconditions. DNA containing the polynucleotide encoding the amylasepolypeptide is then isolated from the host cell, sequenced to identifythe nucleotide sequence changes (if any), and used to express the enzymein a different or the same host cell. Measuring enzyme activity from theexpression libraries can be performed using any suitable method known inthe art (e.g., standard biochemistry techniques, such as HPLC analysis).

For engineered polypeptides of known sequence, the polynucleotidesencoding the enzyme can be prepared by standard solid-phase methods,according to known synthetic methods. In some embodiments, fragments ofup to about 100 bases can be individually synthesized, then joined(e.g., by enzymatic or chemical ligation methods, or polymerase mediatedmethods) to form any desired continuous sequence. For example,polynucleotides and oligonucleotides disclosed herein can be prepared bychemical synthesis using the classical phosphoramidite method (See e.g.,Beaucage et al., Tetra. Lett., 1981, 22:1859-69; and Matthes et al.,EMBO J., 1984, 3:801-05), as it is typically practiced in automatedsynthetic methods. According to the phosphoramidite method,oligonucleotides are synthesized (e.g., in an automatic DNAsynthesizer), purified, annealed, ligated and cloned in appropriatevectors. However, it is not intended that the present invention belimited to any specific method for production of polynucleotides andoligonucleotides, as any suitable method finds use in the presentinvention.

Accordingly, in some embodiments, a method for preparing the engineeredamylase polypeptide can comprise: (a) synthesizing a polynucleotideencoding a polypeptide comprising an amino acid sequence selected fromthe amino acid sequence of any variant provided in Table 4-1, 4-2, 4-3,4-4, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 4-11, 4-12, 4-13, and/or 4-14; anyvariant provided in Table 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7, 4-8, 4-9,4-10, 4-11, 4-12, 4-13, and/or 4-14, wherein the variant lacks thehistidine tag and preceding amino acid linker; as well as SEQ ID NOS: 2,18, 40, 156, 174, 276, 308, 422, 456, 546, 632, 664, 780, 988, 1104,1122, 1224, 1256, 1370, 1404, 1494, 1580, 1612, and/or 1728; any of theeven numbered polypeptide sequence of SEQ ID NOS: 4-18, 22-966, and970-1914; and (b) expressing the amylase polypeptide encoded by thepolynucleotide. In some embodiments of the method, the amino acidsequence encoded by the polynucleotide comprises one or several (e.g.,up to 3, 4, 5, or up to 10) amino acid residue deletions, insertionsand/or substitutions. In some further embodiments, the amino acidsequence comprises 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15,1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45, or 1-50amino acid residue deletions, insertions and/or substitutions. In stillsome other embodiments, the amino acid sequence comprises 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 30, 31, 32, 33, 34, 35, 40, 45, or 50 amino acid residue deletions,insertions and/or substitutions. In some embodiments, the amino acidsequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 18, 20, 21, 22, 23, 24, or 25 amino acid residue deletions,insertions and/or substitutions. In some embodiments, the substitutionsare conservative or non-conservative substitutions.

The expressed engineered amylase polypeptide can be assessed for anydesired improved property (e.g., activity, selectivity, stability, acidtolerance, protease sensitivity, etc.), using any suitable assay knownin the art, including but not limited to the assays and conditionsdescribed herein.

In some embodiments, any of the engineered amylase polypeptidesexpressed in a host cell are recovered from the cells and/or the culturemedium using any one or more of the well-known techniques for proteinpurification, including, among others, lysozyme treatment, sonication,filtration, salting-out, heat treatment, ultra-centrifugation, andchromatography.

Chromatographic techniques for isolation of the amylase polypeptidesinclude, among others, reverse phase chromatography high performanceliquid chromatography, ion exchange chromatography, hydrophobicinteraction chromatography, gel electrophoresis, and affinitychromatography. Conditions for purifying a particular enzyme depends, inpart, on factors such as net charge, hydrophobicity, hydrophilicity,molecular weight, molecular shape, etc., and will be apparent to thosehaving skill in the art. In some embodiments, affinity techniques may beused to isolate the improved variant amylase enzymes. In someembodiments utilizing affinity chromatography purification, any antibodywhich specifically binds the variant amylase polypeptide finds use.

In some embodiments utilizing affinity chromatography purification,proteins that bind to the glycans covalently attached to amylase finduse. In still other embodiments utilizing affinity-chromatographypurifications, any small molecule that binds to the amylase active sitefinds use. For the production of antibodies, various host animals,including but not limited to rabbits, mice, rats, etc., are immunized byinjection with a polypeptide (e.g., a amylase variant), or a fragmentthereof. In some embodiments, the amylase polypeptide or fragment isattached to a suitable carrier, such as BSA, by means of a side chainfunctional group or linkers attached to a side chain functional group.

In some embodiments, the engineered amylase polypeptide is produced in ahost cell by a method comprising culturing a host cell (e.g., E. coli,S. cerevisiae, Daucus carota, Nicotiana tabacum, H. sapiens (e.g.,HEK293T), or Cricetulus griseus (e.g., CHO)) comprising a polynucleotidesequence encoding an engineered amylase polypeptide as described hereinunder conditions conducive to the production of the engineered amylasepolypeptide and recovering the engineered amylase polypeptide from thecells and/or culture medium.

In some embodiments, the invention encompasses a method of producing anengineered amylase polypeptide comprising culturing a recombinanteukaryotic cell comprising a polynucleotide sequence encoding anengineered amylase polypeptide having at least 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to a reference sequence (e.g., SEQ ID NO: 2, 18, 40, 156, 174,276, 308, 422, 456, 546, 632, 664, 780, 988, 1104, 1122, 1224, 1256,1370, 1404, 1494, 1580, 1612, and/or 1728), and one or more amino acidresidue differences as compared to SEQ ID NO: 2, 18, 40, 156, 174, 276,308, 422, 456, 546, 632, 664, 780, 988, 1104, 1122, 1224, 1256, 1370,1404, 1494, 1580, 1612, and/or 1728, selected from those provided inTable 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 4-11, 4-12,4-13, and/or 4-14, and/or combinations thereof when optimally alignedwith the amino acid sequence of SEQ ID NO: 2, 18, 40, 156, 174, 276,308, 422, 456, 546, 632, 664, 780, 988, 1104, 1122, 1224, 1256, 1370,1404, 1494, 1580, 1612, and/or 1728, under suitable culture conditionsto allow the production of the engineered amylase polypeptide andoptionally recovering the engineered amylase polypeptide from theculture and/or cultured bacterial cells.

In some embodiments, once the engineered polypeptides are recovered fromthe recombinant host cells or cell culture medium, they are furtherpurified by any suitable method(s) known in the art. In some additionalembodiments, the purified amylase polypeptides are combined with otheringredients and compounds to provide compositions and formulationscomprising the engineered amylase polypeptide as appropriate fordifferent applications and uses (e.g., pharmaceutical compositions). Insome additional embodiments, the purified polypeptides, or theformulated amylase polypeptides are lyophilized.

Compositions

The present invention provides various compositions and formats,including but not limited to those described below. In some embodiments,the present invention provides engineered amylase polypeptides suitablefor use in pharmaceutical and other compositions, such as dietary and/ornutritional supplements.

Depending on the mode of administration, these compositions comprising atherapeutically effective amount of an engineered amylase according tothe invention are in the form of a solid, semi-solid, or liquid. In someembodiments, the compositions include other pharmaceutically acceptablecomponents such as diluents, buffers, excipients, salts, emulsifiers,preservatives, stabilizers, fillers, and other ingredients. Details ontechniques for formulation and administration are well known in the artand described in the literature.

In some embodiments, the engineered amylase polypeptides are formulatedfor use in pharmaceutical compositions. Any suitable format for use indelivering the engineered amylase polypeptides find use in the presentinvention, including but not limited to pills, tablets, gel tabs,capsules, lozenges, dragees, powders, soft gels, sol-gels, gels,emulsions, implants, patches, sprays, ointments, liniments, creams,pastes, jellies, paints, aerosols, chewing gums, demulcents, sticks,solutions, suspensions (including but not limited to oil-basedsuspensions, oil-in water emulsions, etc.), slurries, syrups, controlledrelease formulations, suppositories, etc. In some embodiments, theengineered amylase polypeptides are provided in a format suitable forinjection or infusion (i.e., in an injectable formulation). In someembodiments, the engineered amylase polypeptides are provided inbiocompatible matrices such as sol-gels, including silica-based (e.g.,oxysilane) sol-gels. In some embodiments, the engineered amylasepolypeptides are encapsulated and/or enterically coated. In somealternative embodiments, the engineered amylase polypeptides areencapsulated in nanostructures (e.g., nanotubes, nanotubules,nanocapsules, or microcapsules, microspheres, liposomes, etc.). Indeed,it is not intended that the present invention be limited to anyparticular delivery formulation and/or means of delivery. It is intendedthat the engineered amylase polypeptides be administered by any suitablemeans known in the art, including but not limited to parenteral, oral,topical, transdermal, intranasal, intraocular, intrathecal, viaimplants, etc.

In some embodiments, the engineered amylase polypeptides are chemicallymodified by glycosylation, chemical crosslinking reagents, pegylation(i.e., modified with polyethylene glycol [PEG] or activated PEG, etc.)or other compounds (See e.g., Ikeda, Amino Acids, 2005, 29:283-287; U.S.Pat. Nos. 7,531,341, 7,534,595, 7,560,263, and 7,553,653; US Pat. Appln.Publ. Nos. 2013/0039898, 2012/0177722, etc.). Indeed, it is not intendedthat the present invention be limited to any particular delivery methodand/or mechanism.

In some additional embodiments, the engineered amylase polypeptides areprovided in formulations comprising matrix-stabilized enzyme crystals.In some embodiments, the formulation comprises a cross-linkedcrystalline engineered amylase enzyme and a polymer with a reactivemoiety that adheres to the enzyme crystals. The present invention alsoprovides engineered amylase polypeptides in polymers.

In some embodiments, compositions comprising the engineered amylasepolypeptides of the present invention include one or more commonly usedcarrier compounds, including but not limited to sugars (e.g., lactose,sucrose, mannitol, and/or sorbitol), starches (e.g., corn, wheat, rice,potato, or other plant starch), cellulose (e.g., methyl cellulose,hydroxypropylmethyl cellulose, sodium carboxy-methylcellulose), gums(e.g., arabic, tragacanth, guar, etc.), and/or proteins (e.g., gelatin,collagen, etc.).

In some embodiments, the engineered amylase polypeptide is suitable foruse to improve dietary starch digestion. In some further embodiments,the engineered amylase polypeptide finds use in breaking down starch infood and feed, prior to the consumption of the food or feed. The dosageof engineered amylase polypeptide(s) administered depends upon thecondition or disease, the general condition of the subject, and otherfactors known to those in the art. In some embodiments, the compositionsare intended for single or multiple administrations. In someembodiments, it is contemplated that the concentration of engineeredamylase polypeptide(s) in the composition(s) administered to a humanwith pancreatic insufficiency disease is sufficient to effectivelytreat, and/or ameliorate disease (e.g., pancreatic insufficiencydisease). In some embodiments, one or more of the engineered amylasepolypeptide(s) of the present disclosure, for example formulated in ascomposition(s), is administered to a human with pancreatic insufficiencydisease in an amount sufficient to effectively treat, and/or amelioratedisease pancreatic insufficiency disease. In some embodiments, theengineered amylase polypeptides are administered in combination withother pharmaceutical and/or dietary compositions. In some embodiments,the engineered amylase polypeptides find use in combination with otherenzymes, such as lipases and/or proteases, for the treatment of diseasessuch as pancreatic enzyme insufficiency. In some embodiments, thesubject is for treatment is a non-human mammal, such as, but not limitedto, a non-human primate, pigs, cows, dogs, and cats.

In some embodiments, the engineered lipase polypeptides are administeredin combination with other pharmaceutical and/or dietary compositions,including but not limited to dietary supplements, nutraceuticals, etc.It is not intended that the present invention be limited to any specificmethod or form of administration, as any suitable method and/or formfinds use.

Examples

The following Examples, including experiments and results achieved, areprovided for illustrative purposes only and are not to be construed aslimiting the present invention. In the experimental disclosure below,the following abbreviations apply: ppm (parts per million); M (molar);mM (millimolar), uM and μM (micromolar); nM (nanomolar); mol (moles); gmand g (gram); mg (milligrams); ug and μg (micrograms); L and 1 (liter);ml and mL (milliliter); cm (centimeters); mm (millimeters); um and μm(micrometers); sec. (seconds); min(s) (minute(s)); h(s) and hr(s)(hour(s)); kDa (kilodaltons); U (units); MW (molecular weight); rpm(rotations per minute); ° C. (degrees Centigrade); CDS (codingsequence); DNA (deoxyribonucleic acid); RNA (ribonucleic acid); E. coliW3110 (commonly used laboratory E. coli strain, available from the ColiGenetic Stock Center [CGSC], New Haven, Conn.); EPI (exocrine pancreaticinsufficiency); ethylidene-pNP-G7(ethylidene-4-nitrophenyl-alpha-D-maltoheptaoside); btLIP (B.thermoamylovorans lipase); HPLC (high pressure liquid chromatography);ms (mass spectrometry or mass spectroscopy); SDS-PAGE (sodium dodecylsulfate polyacrylamide gel electrophoresis); PBS (phosphate bufferedsaline); PES (polyethersulfone); ACN (acetonitrile); DNSA(dinitrosalicylic acid); IPA (isopropyl alcohol); IPTG (isopropylβ-D-1-thiogalactopyranoside); PMBS (polymyxin B sulfate); NADPH(nicotinamide adenine dinucleotide phosphate); LB (Luria broth); MeOH(methanol); FIOPC and FIOP (fold improvements over positive control);HTP (high throughput); CAV (cell accelerator voltage; collision cellaccelerator voltage); CE (collision energy); RF (radio frequency); NEB(New England Biolabs, Inc., Ipswich, Mass.); Biorad (Bio-RadLaboratories, Inc., Hercules, Calif.); Greiner (Greiner Bio-One NorthAmerica, Inc., Monroe, N.C.); GE Healthcare (GE Healthcare, Chicago,Ill.); Sigma-Aldrich (Sigma-Aldrich, St. Louis, Mo.); Pall Corp. (Pall,Corp., Pt. Washington, N.Y.); Millipore (Millipore, Corp., BillericaMass.); Difco (Difco Laboratories, BD Diagnostic Systems, Detroit,Mich.); Molecular Devices (Molecular Devices, LLC, Sunnyvale, Calif.);Kuhner (Adolf Kuhner, A G, Basel, Switzerland); Applied Biosystems(Applied Biosystems, part of Life Technologies, Corp., Grand Island,N.Y.), Agilent (Agilent Technologies, Inc., Santa Clara, Calif.);RAPIDFIRE® MS (RAPIDFIRE® mass spectrometer, Agilent); Thermo Scientific(part of ThermoFisher Scientific, Waltham, Mass.); Gibco (ThermoFisherScientific); Pierce (Pierce Biotechnology (now part of Thermo FisherScientific), Rockford, Ill.); ThermoFisher Scientific (Thermo FisherScientific, Waltham, Mass.); Corning (Corning, Inc., Palo Alto, Calif.);and Bio-Rad (Bio-Rad Laboratories, Hercules, Calif.).

Example 1—Amylase Gene Acquisition and Construction of ExpressionVectors

The DNA sequences encoding bacterial amylases from various organismslisted in Table 1-1, were codon-optimized for expression in E. coli, andcloned into the E. coli expression vector pCK110900 vector system (Seee.g., U.S. Pat. Nos. 7,629,157, and 9,714,437, and US Pat. Appln. Publn.2006/0195947, all of which are hereby incorporated by reference herein).The plasmid construct was transformed into an E. coli strain derivedfrom W3110. The E. coli strains containing the amylase genes were grownin either 96-well or shake flask format and assayed for amylase activityas described in Example 3. The activity obtained from these amylases areshown in Table 1-1.

TABLE 1-1 Amylase Activity of Alpha-Amylases from Various Organisms¹ SEQID NO: Accession Activity (nt/aa) No. Organism (A405) 1/2 P26612Escherichia +++ coli 3/4 CAA44638.1 Alicyclobacillus + acidocaldarius5/6 AAF00567.1 Cytophaga sp. +++ 7/8 P26613 Salmonella +++ typhimurium 9/10 ADD77452.1 Pantoea + ananatis 11/12 WP_004392868.1 Yersinia +++kristensenii 13/14 WP_017372073.1 Enterobacteriaceae + bacterium LSJC715/16 WP_059351662.1 Bacillus ++ coahuilensis ¹Levels of activity areexpressed as absorbance units at 405 nm and defined as follows: “+” >0.004; “++” > 0.04; and “+++” > 0.4.

Example 2—Engineered Amylase Variants Expression and Construction

Based on the results shown in Table 1-1, the amylase from SEQ ID NO: 2was subcloned with a C-terminal 6×His-tag into the pJV110900 vectorsystem (See e.g., US Pat. Appln Publ. 2017/213758, hereby incorporatedby reference) to generate SEQ ID NO: 18. However, it is not intendedthat the present invention be limited to any specific vectors. Theplasmid construct was transformed into an E. coli strain derived fromW3110. Directed evolution techniques generally known by those skilled inthe art were used to generate libraries of gene variants from thisplasmid construct (See e.g., U.S. Pat. No. 8,383,346, and WO2010/144103)as well as its derivatives.

Example 3—High-Throughput (HTP) Growth of Amylase Variants and ScreeningConditions

Methods used to grow cells producing the amylases of the presentinvention, as well as screening methods to characterize the amylases areprovided in this Example.

High-Throughput (HTP) Growth of E. coli Amylase and Variant Amylases

Transformed E. coli cells were selected by plating onto LB agar platescontaining 1% glucose with selection. After overnight incubation at 37°C., colonies were placed into the wells of 96-well shallow flat bottomplates (NUNC™, Thermo-Scientific) filled with 180 μl/well LBsupplemented with 1% glucose and a selection marker. The cultures wereallowed to grow overnight for 18-20 hours in a shaker incubator (200rpm, 30° C., and 85% relative humidity; Kuhner).

Overnight growth samples (20 μL) were transferred into COSTAR® 96-welldeep plates filled with 380 μL of Terrific Broth supplemented with aselection compound (e.g., chloramphenicol). The plates were incubatedfor 135 minutes in a shaker incubator (250 rpm, 30° C., and 85% relativehumidity; Kuhner). The expression of the amylase variants was theninduced with 40 μL of 10 mM IPTG in sterile water and the cultures wereincubated overnight for 20-24 hours in a shaker incubator (250 rpm, 30°C., and 85% relative humidity; Kuhner). The cells were pelleted bycentrifugation (4000 rpm×20 min), the supernatants were discarded, andthe cells were frozen at −80° C. prior to analysis.

Lysis of HTP Pellets

First, 200-400 μL of lysis buffer (1×PBS, 1 mg/ml lysozyme, 0.5 mg/mlpolymyxin B sulfate, and 0.1U/mL DNAse I (NEB) were added to the cellpellets. The cell pellet and buffer were gently shaken for 1.5-2 hoursat room temperature, and centrifuged (4000 rpm×15 min) prior to use ofthe clarified lysates in the various HTP assays described herein.Analysis of these lysates by SDS-PAGE revealed the presence of anoverexpressed protein bands at an apparent MW of 57 kDa, consistent withthe expected MW of the amylase of interest. In some additionalexperiments, additional dilutions of clarified lysate were performedwith PBS buffer prior to challenges and analysis.

Analysis of Clarified Lysates or Purified Protein for Amylase Activity

Amylase activity was assessed using soluble starch as a substrate andquantifying maltose produced from starch hydrolysis usingdinitrosalicylic acid (DNSA). Starch solution was prepared by mixing 10mg/mL potato starch in assay buffer (20 mM sodium phosphate buffer pH6.9+6.7 mM NaCl, or 100 mM NaPO₄ pH, 6.0-7.0). To dissolve the starch,the solution was heated on a stir plate to near boiling then allowed tocool to room temperature. To create the DNSA solution, 20 ml of 96 mMDNSA in water was added dropwise to 12 ml water and 8 ml 5.3M potassiumsodium tartrate tetrahydrate in 2N NaOH with mixing on a hot plate(70-80° C.); DNSA solution was cooled to room temperature before use. Toset up amylase activity reactions, starch substrate solution was mixed2:1 with diluted enzyme in a PCR plate (BioRad) and incubated at 37° C.for 30 minutes. The reaction was then mixed 1:1 with the DNSA solutionand incubated for 15 minutes at 95° C. in a thermocycler. Samples werediluted 2× in assay buffer and transferred to a UV-STAR® flat bottomplate (Greiner), then read for absorbance at 540 nm on a MolecularDevices SPECTRAMAX® M2 plate reader. In some experiments, in order toensure that samples did not saturate the assay, samples were diluted inPBS buffer prior to analysis, based on a pre-determined dilution factor(up to 512×).

Alternate Method for Analysis of Clarified Lysates or Purified Proteinfor Amylase Activity

In some experiments, amylase activity was assessed by using ethylidenepNP-G7 as a substrate and monitoring the p-nitrophenol released bymonitoring absorbance at A405. This assay was performed using an AmylaseActivity Assay Kit (Sigma). In this assay, 50 ul of challenged, dilutedamylase variant were added to 50 ul of provided assay buffer and 50 ulof provided ethylidene pNP-G7 substrate solution. Absorbance at 405 nmwas monitored for 15 minutes, and absorbance at a representative timepoint was used for subsequent analysis.

HTP Analysis of Clarified Lysates or Purified Proteins Pre-Treated withHeat Challenge

To assess the thermostability of the variants, clarified lysate wastransferred to a PCR plate (Biorad) and incubated for 1 hr at 40-60° C.in a thermocycler. After incubation, samples were centrifuged to pelletheat-precipitated proteins and the supernatant was analyzed for amylaseresidual activity as described herein.

HTP Analysis of Clarified Lysates or Purified Proteins Pre-Treated withlow pH and Pepsin

The activities of amylase variants were determined after pre-incubationat low pH in the presence of pepsin, to simulate the stomachenvironment. First, clarified lysate was diluted 0×-32× in PBS buffer,then mixed 1:1 with McIlvaine buffer, pH 2.3-3.5+3.0 mg/mL pepsin fromporcine gastric mucosa (Sigma) in a PCR plate (Biorad), for a finalchallenge pH of 2.3-3.5 and a final pepsin concentration of 1.5 mg/ml.Samples were mixed and then incubated for 1-2 hrs at 37° C. To stop thechallenges, samples were mixed 1:1 with 400 mM NaPO₄, pH 7.0, toneutralize the pH and inactivate the pepsin. Neutralized challengesamples were then analyzed for residual amylase activity as describedherein.

HTP Evaluation of Amylase Activity After Incubation with IntestinalProteases

The activities of variants were determined after pre-incubation atneutral pH in the presence of sodium taurocholate and intestinalproteases, to simulate the intestinal environment. Clarified lysate wasmixed 1:1 in a PCR plate (BioRad) with 100 mM NaPO₄, pH 7.0, or PBScontaining 0-10 mM sodium taurocholate, trypsin from porcine pancreas at3 mg/ml, and chymotrypsin from bovine pancreas at 3 mg/ml, for a finalconcentration of 0-5 mM sodium taurocholate, and a final concentrationof 1.5 mg/ml of each challenging intestinal protease. The mixture wasincubated for 1-2 hrs at 37° C., in a thermocycler. Samples were thenanalyzed for residual amylase activity as described herein.

HTP Evaluation of Amylase Activity After Incubation with EngineeredProtease

The activities of amylase variants were determined after pre-incubationat neutral pH in the presence of an engineered trypsin-like protease(SEQ ID NO: 20). Clarified lysate was mixed 1:1 in a PCR plate (BioRad)with PBS containing 3 mg/ml lyophilized powder of SEQ ID NO: 20, for afinal concentration of 1.5 mg/ml protease in the challenge. The mixturewas incubated for 1-2 hrs at 37° C., in a thermocycler. Samples werethen analyzed for residual amylase activity as described herein.

HTP Evaluation of Amylase Activity After Multiple Sequential Challenges

When two stability characteristics were simultaneously beinginvestigated, multiple challenge screens were combined in series. Forexample, in some experiments, a one hour heat challenge was followed bya one hour pH and pepsin challenge, which was then followed by a onehour intestinal challenge. In the instance of an intestinal challengefollowing a gastric challenge, the challenging intestinal proteases weredissolved in the neutralization buffer for the gastric challenge, sothat the intestinal challenge was started immediately uponneutralization of the gastric challenge. This is denoted as a“sequential challenge.” Following a sequential challenge, samples wereanalyzed for residual amylase activity as described herein.

Shake Flask Expression, Purification and Characterization of AmylaseVariants

A single colony of E. coli containing a plasmid encoding an engineeredamylase was inoculated into 5 mL Luria Bertani media with 1% glucose anda selection marker. Cells were grown overnight (at least 16 hours) in anincubator at 30° C. with shaking at 250 rpm. The culture was dilutedinto 250 mL Terrific Broth (12 g/L bacto-tryptone, 24 g/L yeast extract,4 mL/L glycerol, 65 mM potassium phosphate, pH 7, 1 mM MgSO₄) withselection, in a 1 liter flask and allowed to grow at 30° C. Expressionof the amylase gene was induced by addition of IPTG to a finalconcentration of 1 mM when the OD600 of the culture reached 0.6 to 0.8.Incubation was then continued overnight (at least 16 hours). Cells wereharvested by centrifugation (5000 rpm, 15 min, 4° C.) and thesupernatant discarded. The cells were resuspended in 40-50 ml of PBSwith 150 mM NaCl, and passed through a MICROFLUDIZER® high pressurehomogenizer (Microfluidics) with standard E. coli lysis settings andmaintained at 4° C.

The cleared lysate supernatant was collected, and imidazole was added tothe lysate for a final concentration of 25 mM. This lysate wasHIS-affinity purified on the AKTA Start system with the AC Step 1 ml/5ml HiTrap—Affinity Chromatography Quick Start using the manufacturer's(GE Healthcare) protocol, using PBS with 150 mL NaCl and 25 mM imidazoleas the wash buffer, and PBS with 150 ml NaCl and 250 mM imidazole as theelution buffer. The purified amylase variants were buffer exchanged intoPBS with ECONOPAC®-10DG desalting prepacked gravity-flow columns(Biorad).

The purified amylase variants were assayed for unchallenged activity,gastric stability, intestinal stability in a similar manner as describedherein, except that instead of using cell lysates, 1 mg/ml of thepurified enzymes were used in the stability experiments.

Example 4: Screening Results For Amylase Variants

Library variants were generated from homolog diversity and saturationmutagenesis on surface positions based on a structural model of SEQ IDNO: 18. These variants were screened in triplicate for amylase activityat pH 6.8, after the sequential challenge of 1 hour incubation at 50°C., followed by a 1 hour gastric challenge (1.5 mg/mL pepsin, pH 3), andfinally a 1 hour intestinal challenge for 1 hour, as described inExample 3. The results (relative to the results for SEQ ID NO: 18) areprovided in Table 4-1.

TABLE 4-1 Amylase Activity (Relative to SEQ ID NO: 18)¹ Amino AcidAmylase Activity Post Differences Sequential Challenge SEQ ID NO:(Relative to (Relative to SEQ ID (nt/aa) SEQ ID NO: 18) NO: 18) 21/22K158R ++++ 23/24 D396H/L412D ++++ 25/26 K284S ++++ 27/28 E26A/R27L/L412D++++ 29/30 D272G +++ 31/32 P74A/Y371F/H374S/ +++ Q383E/E493D 33/34E26A/R27L/Q466E/E473D +++ 35/36 V317G/Q383L +++ 37/38D396H/L412D/D428E/Q466E +++ 39/40 E16D/D33E/F157Y/A256S/ +++M302Q/Y371F/Q383E 41/42 P257G +++ 43/44 D388E/D396H/L412D/D428E +++45/46 E26A/R27L/D396H/L412D/ +++ D428E/E473D 47/48 Q149R +++ 49/50A256S/Q383E +++ 51/52 E130D/A256S/E493D +++ 53/54 P257E +++ 55/56E16D/A256S/M302Q/H374S +++ 57/58 I73V/E130D/A256S/E282D/ ++ M302Q/E493D59/60 D272K ++ 61/62 E16D/I73V/P74A/A256S/ ++ E282D/F366L/Y371F/Q383E/E493D 63/64 L412S ++ 65/66 K284R ++ 67/68 E16D/I73V/A256S/E282D/ ++M302Q/H331P/Y371F/Q383E 69/70 D272S ++ 71/72 D29R ++ 73/74 D272P ++75/76 P257R ++ 77/78 D279R ++ 79/80 D272R ++ 81/82 K284C ++ 83/84E16D/E130D/A256S/T276Q/ ++ H374S/V470T 85/86 P257S ++ 87/88 V409S ++89/90 E254S ++ 91/92 I495E ++ 93/94 K19E/S72T/P345A/E473D ++ 95/96 P257V++ 97/98 D29S ++  99/100 E16D/D33E/E130D/A256S/ ++M302Q/Y371F/Q383E/V470T 101/102 K284Y ++ 103/104 A256S ++ 105/106 E254G++ 107/108 R2K/I73V/A256S/E493D ++ 109/110 E16D/D33E/I73V/E130D/ ++A256S/E282D/Y371F/H374S/ E493D 111/112 Q149T ++ 113/114P345A/D388E/D396H/D428E ++ 115/116 I495L ++ 117/118 F322A + 119/120D29G + 121/122 P259Q + 123/124 I495Y + ¹Levels of increased activitywere determined relative to the reference polypeptide of SEQ ID NO: 18,and defined as follows: “+” > 0.9; “++” > 1.1; “+++” > 2; and “++++” >4.

Based on the results shown in Table 4-1, SEQ ID NO: 40 was chosen as theparent sequence for the next iteration of protein optimization.Beneficial mutations identified from those listed in Table 4-2 wererecombined into this backbone. The variants were assayed in triplicatefor amylase activity at pH 6.8 after pre-incubations with two differentconditions: a 1 hr incubation at 50° C., followed by a 1 hr intestinalscreen, and a one hour incubation at 50° C., followed by a 1 hr gastricchallenge at pH 3.5, as described in Example 3. The results (relative tothe results for SEQ ID NO: 40) are shown in Table 4-2.

TABLE 4-2 Alpha-Amylase Activity (Relative to SEQ ID NO: 40)¹ AmylaseActivity Amylase Activity Post Heat & Post Heat & Amino Acid Amino AcidIntestinal Gastric Differences Differences Challenge Challenge SEQ IDNO: (Relative to (Relative to (Relative to SEQ (Relative to SEQ (nt/aa)SEQ ID NO: 40) SEQ ID NO: 18) ID NO: 40) ID NO: 40) 125/126D272G/D279R/L412D/ E16D/D33E/F157Y/A256S/ +++ ++++ D428E/E493DD272G/D279R/M302Q/Y371F/ Q383E/L412D/D428E/E493D 127/128D272G/L412D/D428E E16D/D33E/F157Y/A256S/ +++ +++D272G/M302Q/Y371F/Q383E/ L412D/D428E 129/130 D272G/L412D/E493DE16D/D33E/F157Y/A256S/ +++ +++ D272G/M302Q/Y371F/Q383E/ L412D/E493D131/132 D29R/D272G/D279R/ E16D/D29R/D33E/F157Y/ +++ +++ D396H/L412DA256S/D272G/D279R/M302Q/ Y371F/Q383E/D396H/L412D 133/134E130D/E254S/K284S/ E16D/D33E/E130D/F157Y/ +++ +++ F322A/H374S/V409S/E254S/A256S/K284S/M302Q/ Q466E F322A/Y371F/H374S/Q383E/ V409S/Q466E135/136 E130D/K284S/F322A/ E16D/D33E/E130D/F157Y/ +++ ++H374S/V409S/E473D A256S/K284S/M302Q/F322A/ Y371F/H374S/Q383E/V409S/E473D 137/138 E130D/P257G/K284S/ E16D/D33E/E130D/F157Y/ +++ +++H374S/V409S/Q466E A256S/P257G/K284S/M302Q/ Y371F/H374S/Q383E/V409S/Q466E 139/140 E130D/Q149R/E254S/ E16D/D33E/E130D/Q149R/ +++ +++K284S/F322A/V409S/ F157Y/E254S/A256S/K284S/ I495EM302Q/F322A/Y371F/Q383E/ V409S/I495E 141/142 E130D/Q149R/P257G/E16D/D33E/E130D/Q149R/ +++ +++ K284S/F322A/V409S/F157Y/A256S/P257G/K284S/ Q466E/E473D M302Q/F322A/Y371F/Q383E/V409S/Q466E/E473D 143/144 E254S/K284S/V409S/ E16D/D33E/F157Y/E254S/ +++++ Q466E/I495E A256S/K284S/M302Q/Y371F/ Q383E/V409S/Q466E/I495E 145/146E254S/P257G/K284S/ E16D/D33E/F157Y/E254S/ +++ ++ F322A/V409S/E473DA256S/P257G/K284S/M302Q/ F322A/Y371F/Q383E/V409S/ E473D 147/148E26A/D272G/D279R/ E16D/E26A/D33E/F157Y/ +++ ++++ L412D/D428E/E493DA256S/D272G/D279R/M302Q/ Y371F/Q383E/L412D/D428E/ E493D 149/150E26A/D29R/D272G/ E16D/E26A/D29R/D33E/ +++ +++ D279R/L412D/E493DF157Y/A256S/D272G/D279R/ M302Q/Y371F/Q383E/L412D/ E493D 151/152E26A/D29R/D272G/ E16D/E26A/D29R/D33E/ ++ +++ D396H/L412DF157Y/A256S/D272G/M302Q/ Y371F/Q383E/D396H/L412D 153/154E26A/D29R/D279R/ E16D/E26A/D29R/D33E/ +++ +++ L412D/D428EF157Y/A256S/D279R/M302Q/ Y371F/Q383E/L412D/D428E 155/156E26A/D29R/K158R/ E16D/E26A/D29R/D33E/ +++ +++ D272G/L412DF157Y/K158R/A256S/D272G/ M302Q/Y371F/Q383E/L412D 157/158E26A/K158R/D272G/ E16D/E26A/D33E/F157Y/ +++ +++ D279R/D388E/L412D/K158R/A256S/D272G/D279R/ D428E M302Q/Y371F/Q383E/D388E/ L412D/D428E159/160 E26A/K158R/D272G/ E16D/E26A/D33E/F157Y/ +++ +++ L412DK158R/A256S/D272G/M302Q/ Y371F/Q383E/L412D 161/162 K158R/D272G/D279R/E16D/D33E/F157Y/K158R/ +++ +++ Q394R/D396H/L412D/A256S/D272G/D279R/M302Q/ D428E/E493D Y371F/Q383E/Q394R/D396H/L412D/D428E/E493D 163/164 K158R/D272G/D388E/ E16D/D33E/F157Y/K158R/ ++++++ L412D/D428E/E493D A256S/D272G/M302Q/Y371F/ Q383E/D388E/L412D/D428E/E493D 165/166 K158R/D272G/L412D/ E16D/D33E/F157Y/K158R/ ++ +++ D428EA256S/D272G/M302Q/Y371F/ Q383E/L412D/D428E 167/168 K158R/D279R/D388E/E16D/D33E/F157Y/K158R/ ++ +++ D396H/L412D/D428E A256S/D279R/M302Q/Y371F/Q383E/D388E/D396H/L412D/ D428E 169/170 K284S/F322A/E16D/D33E/F157Y/K284S/ +++ ++ V409S/E473D A256S/M302Q/F322A/Y371F/Q383E/V409S/E473D 171/172 Q149R/P257G/K284S/ E16D/D33E/Q149R/F157Y/ ++++++ F322A/V409S/Q466E A256S/P257G/K284S/M302Q/ F322A/Y371F/Q383E/V409S/Q466E ¹Levels of increased activity were determined relative to thereference polypeptide of SEQ ID NO: 40, and defined as follows: “+” >0.9; “++” > 1.1; “+++” > 2; and “++++” > 3.

Based on the results shown in Table 4-2, SEQ ID NO: 156 was chosen asthe parent sequence for the next iteration of protein optimization.Beneficial mutations identified from the results shown in Table 4-2 wererecombined into the backbone. Additionally, to generate new diversity,variants produced with saturation mutagenesis at different positionswere also constructed using SEQ ID NO: 156.

The variants were assayed in triplicate for amylase activity at pH 6.8,after pre-incubation under two different conditions. The first conditionwas a 1 hour gastric challenge at pH 3.25 and the second condition was a2 hr gastric challenge at pH 3.25, as described in Example 3. Theresults (relative to the results for SEQ ID NO: 156) are provided inTable 4-3.

TABLE 4-3 Alpha-Amylase Activity (Relative to SEQ ID NO: 156)¹ AmylaseActivity Amylase Activity Post 1 Hour Post 2 Hour Amino Acid Amino AcidGastric Gastric SEQ ID Differences Differences Challenge Challenge NO:(Relative to (Relative to (Relative to (Relative to (nt/aa) SEQ ID NO:156) SEQ ID NO: 18) SEQ ID NO: 156) SEQ ID NO: 156) 173/174 A114HE16D/E26A/D29R/D33E/A114H/F157Y/ +++ +++ K158R/A256S/D272G/M302Q/Y371F/Q383E/L412D 175/176 K91H E16D/E26A/D29R/D33E/K91H/F157Y/ +++ +++K158R/A256S/D272G/M302Q/Y371F/ Q383E/L412D 177/178 A122PE16D/E26A/D29R/D33E/A122P/F157Y/ ++ ++ K158R/A256S/D272G/M302Q/Y371F/Q383E/L412D 179/180 E167W E16D/E26A/D29R/D33E/F157Y/K158R/ ++ ++E167W/A256S/D272G/M302Q/Y371F/ Q383E/L412D 181/182 G183NE16D/E26A/D29R/D33E/F157Y/K158R/ ++ ++ G183N/A256S/D272G/M302Q/Y371F/Q383E/L412D 183/184 A122R E16D/E26A/D29R/D33E/A122R/F157Y/ ++ ++K158R/A256S/D272G/M302Q/Y371F/ Q383E/L412D 185/186 N464LE16D/E26A/D29R/D33E/F157Y/K158R/ ++ + A256S/D272G/M302Q/Y371F/Q383E/L412D/N464L 187/188 G183E E16D/E26A/D29R/D33E/F157Y/K158R/ ++ ++G183E/A256S/D272G/M302Q/Y371F/ Q383E/L412D 189/190 R140KE16D/E26A/D29R/D33E/R140K/F157Y/ ++ + K158R/A256S/D272G/M302Q/Y371F/Q383E/L412D 191/192 E397T E16D/E26A/D29R/D33E/F157Y/K158R/ ++ ++A256S/D272G/M302Q/Y371F/Q383E/ E397T/L412D 193/194 D124RE16D/E26A/D29R/D33E/D124R/F157Y/ ++ ++ K158R/A256S/D272G/M302Q/Y371F/Q383E/L412D 195/196 K112Q E16D/E26A/D29R/D33E/K112Q/F157Y/ ++ ++K158R/A256S/D272G/M302Q/Y371F/ Q383E/L412D 197/198 I128WE16D/E26A/D29R/D33E/I128W/F157Y/ ++ ++ K158R/A256S/D272G/M302Q/Y371F/Q383E/L412D 199/200 G51R E16D/E26A/D29R/D33E/G51R/F157Y/ ++ ++K158R/A256S/D272G/M302Q/Y371F/ Q383E/L412D 201/202 T276AE16D/E26A/D29R/D33E/F157Y/K158R/ ++ ++ A256S/D272G/T276A/M302Q/Y371F/Q383E/L412D 203/204 E111A E16D/E26A/D29R/D33E/E111A/F157Y/ ++ +K158R/A256S/D272G/M302Q/Y371F/ Q383E/L412D 205/206 E298ME16D/E26A/D29R/D33E/F157Y/K158R/ ++ + A256S/D272G/E298M/M302Q/Y371F/Q383E/L412D 207/208 N37R E16D/E26A/D29R/D33E/N37R/F157Y/ ++ +K158R/A256S/D272G/M302Q/Y371F/ Q383E/L412D 209/210 G183VE16D/E26A/D29R/D33E/F157Y/K158R/ + + G183V/A256S/D272G/M302Q/Y371F/Q383E/L412D 211/212 Q280L E16D/E26A/D29R/D33E/F157Y/K158R/ + +A256S/D272G/Q280L/M302Q/Y371F/ Q383E/L412D 213/214 L194RE16D/E26A/D29R/D33E/F157Y/K158R/ + ++ L194R/A256S/D272G/M302Q/Y371F/Q383E/L412D 215/216 T276K E16D/E26A/D29R/D33E/F157Y/K158R/ + ++A256S/D272G/T276K/M302Q/Y371F/ Q383E/L412D 217/218 E171IE16D/E26A/D29R/D33E/F157Y/K158R/ + ++ E171I/A256S/D272G/M302Q/Y371F/Q383E/L412D 219/220 L194E E16D/E26A/D29R/D33E/F157Y/K158R/ + ++L194E/A256S/D272G/M302Q/Y371F/ Q383E/L412D 221/222 E298KE16D/E26A/D29R/D33E/F157Y/K158R/ + ++ A256S/D272G/E298K/M302Q/Y371F/Q383E/L412D ¹Levels of increased activity were determined relative tothe reference polypeptide of SEQ ID NO: 156, and defined as follows:“+” > 0.9; “++” > 1.1; and “+++” > 2.

Based on the results shown in Table 4-3, SEQ ID NO: 174 was chosen asthe parent sequence for the next iteration of protein optimization.Beneficial mutations identified from Table 4-2 and Table 4-3, anddiversity from homologs, were recombined into this backbone. Thevariants were assayed in singlicate for amylase activity at pH 6.8 afterpre-incubation with a 1 hour gastric challenge at pH 3.25, as describedin Example 3. The results (relative to the results for SEQ ID NO: 174)are provided in Table 4-4.

TABLE 4-4 Alpha-Amylase Activity (Relative to SEQ ID NO: 174)¹ AmylaseActivity Amino Acid Amino Acid Post Gastric SEQ ID DifferencesDifferences Challenge NO: (Relative to (Relative to (Relative to (nt/aa)SEQ ID NO: 174) SEQ ID NO: 18) SEQ ID NO: 174) 223/224 A122P/V409SE16D/E26A/D29R/D33E/A114H/A122P/F157Y/K158R/ ++A256S/D272G/M302Q/Y371F/Q383E/V409S/L412D 225/226 A122R/E130D/E171I/E16D/E26A/D29R/D33E/A114H/A122R/E130D/F157Y/ ++ F322A/V409SK158R/E171I/A256S/D272G/M302Q/F322A/Y371F/ Q383E/V409S/L412D 227/228A122R/I128W E16D/E26A/D29R/D33E/A114H/A122R/I128W/F157Y/ ++K158R/A256S/D272G/M302Q/Y371F/Q383E/L412D 229/230 A122R/I128W/L194R/E16D/E26A/D29R/D33E/A114H/A122R/I128W/F157Y/ ++ V409SK158R/L194R/A256S/D272G/M302Q/Y371F/Q383E/ V409S/L412D 231/232A145G/T227L/K295N/ E16D/E26A/D29R/D33E/A114H/A145G/F157Y/K158R/ ++ C481VT227L/A256S/D272G/K295N/M302Q/Y371F/Q383E/ L412D/C481V 233/234A145G/T309S/C481V E16D/E26A/D29R/D33E/A114H/A145G/F157Y/K158R/ ++A256S/D272G/M302Q/T309S/Y371F/Q383E/L412D/ C481V 235/236 F322AE16D/E26A/D29R/D33E/A114H/F157Y/K158R/A256S/ ++D272G/M302Q/F322A/Y371F/Q383E/L412D 237/238 F322A/V409SE16D/E26A/D29R/D33E/A114H/F157Y/K158R/A256S/ ++D272G/M302Q/F322A/Y371F/Q383E/V409S/L412D 239/240 F480T/C481VE16D/E26A/D29R/D33E/A114H/F157Y/K158R/A256S/ ++D272G/M302Q/Y371F/Q383E/L412D/F480T/C481V 241/242 G137A/A145G/K295NE16D/E26A/D29R/D33E/A114H/G137A/A145G/F157Y/ ++K158R/A256S/D272G/K295N/M302Q/Y371F/Q383E/ L412D 243/244G137A/A145G/T227L/ E16D/E26A/D29R/D33E/A114H/G137A/A145G/F157Y/ ++Q394E/F480P/C481V K158R/T227L/A256S/D272G/M302Q/Y371F/Q383E/Q394E/L412D/F480P/C481V 245/246 G183NE16D/E26A/D29R/D33E/A114H/F157Y/K158R/G183N/ ++A256S/D272G/M302Q/Y371F/Q383E/L412D 247/248 K295N/Q394EE16D/E26A/D29R/D33E/A114H/F157Y/K158R/A256S/ ++D272G/K295N/M302Q/Y371F/Q383E/Q394E/L412D 249/250 K91HE16D/E26A/D29R/D33E/K91H/A114H/F157Y/K158R/ ++A256S/D272G/M302Q/Y371F/Q383E/L412D 251/252 K91H/A122PE16D/E26A/D29R/D33E/K91H/A114H/A122P/F157Y/ +++K158R/A256S/D272G/M302Q/Y371F/Q383E/L412D 253/254 K91H/A122P/E130D/E16D/E26A/D29R/D33E/K91H/A114H/A122P/E130D/ +++ G183N/L194RF157Y/K158R/G183N/L194R/A256S/D272G/M302Q/ Y371F/Q383E/L412D 255/256K91H/A122P/E130D/ E16D/E26A/D29R/D33E/K91H/A114H/A122P/E130D/ ++L194R/F322A F157Y/K158R/L194R/A256S/D272G/M302Q/F322A/ Y371F/Q383E/L412D257/258 K91H/A122R E16D/E26A/D29R/D33E/K91H/A114H/A122R/F157Y/ +++K158R/A256S/D272G/M302Q/Y371F/Q383E/L412D 259/260 K91H/A122R/E130D/E16D/E26A/D29R/D33E/K91H/A114H/A122R/E130D/ +++ E167W/G183N/L194R/F157Y/K158R/E167W/G183N/L194R/A256S/D272G/ V409SM302Q/Y371F/Q383E/V409S/L412D 261/262 K91H/A122R/E130D/E16D/E26A/D29R/D33E/K91H/A114H/A122R/E130D/ +++ E171I/G183NF157Y/K158R/E171I/G183N/A256S/D272G/M302Q/Y 371F/Q383E/L412D 263/264K91H/A122R/E130D/ E16D/E26A/D29R/D33E/K91H/A114H/A122R/ +++ L194RE130D/F157Y/K158R/L194R/A256S/D272G/M302Q/ Y371F/Q383E/L412D 265/266K91H/A122R/E167W/ E16D/E26A/D29R/D33E/K91H/A114H/A122R/F157Y/ +++E171I/L194R K158R/E167W/E171I/L194R/A256S/D272G/M302Q/ Y371F/Q383E/L412D267/268 K91H/A122R/E167W/ E16D/E26A/D29R/D33E/K91H/A114H/A122R/F157Y/+++ E171I/L194R/F322A K158R/E167W/E1711/L194R/A256S/D272G/M302Q/F322A/Y371F/Q383E/L412D 269/270 K91H/A122R/E171I/E16D/E26A/D29R/D33E/K91H/A114H/A122R/F157Y/ +++ G183N/L194R/V409SK158R/E171I/G183N/L194R/A256S/D272G/M302Q/ Y371F/Q383E/V409S/L412D271/272 K91H/A122R/I128W/ E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/+++ E130D/E167W/E171I/ E130D/F157Y/K158R/E167W/E171I/A256S/D272G/ V409SM302Q/Y371F/Q383E/V409S/L412D 273/274 K91H/A122R/I128W/E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/ +++ E130D/F322AE130D/F157Y/K158R/A256S/D272G/M302Q/F322A/ Y371F/Q383E/L412D 275/276K91H/A122R/I128W/ E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/ +++E130D/G183N/F322A E130D/F157Y/K158R/G183N/A256S/D272G/M302Q/F322A/Y371F/Q383E/L412D 277/278 K91H/A122R/I128W/E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/ +++ E130D/L194RE130D/F157Y/K158R/L194R/A256S/D272G/M302Q/ Y371F/Q383E/L412D 279/280K91H/A122R/I128W/ E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/ +++E171I/G183N/L194R/ F157Y/K158R/E171I/G183N/L194R/A256S/D272G/ F322AM302Q/F322A/Y371F/Q383E/L412D 281/282 K91H/A122R/I128W/E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/ ++ G183N/V409SF157Y/K158R/G183N/A256S/D272G/M302Q/Y371F/ Q383E/V409S/L412D 283/284K91H/E130D/E171I/ E16D/E26A/D29R/D33E/K91H/A114H/E130D/F157Y/ ++G183N/F322A/V409S K158R/E171I/G183N/A256S/D272G/M302Q/F322A/Y371F/Q383E/V409S/L412D 285/286 K91H/E167W/E171IE16D/E26A/D29R/D33E/K91H/A114H/F157Y/K158R/ +++E167W/E1711/A256S/D272G/M302Q/Y371F/Q383E/ L412D 287/288K91H/E167W/F322A E16D/E26A/D29R/D33E/A114H/F157Y/K158R/A256S/ ++D272G/M302Q/Y371F/Q383E/L412D/ K91H/E167W/F322A 289/290K91H/E167W/G183N/ E16D/E26A/D29R/D33E/K91H/A114H/F157Y/K158R/ +++ L194RE167W/G183N/L194R/A256S/D272G/M302Q/Y371F/ Q383E/L412D 291/292K91H/F322A E16D/E26A/D29R/D33E/K91H/A114H/F157Y/K158R/ ++A256S/D272G/M302Q/F322A/Y371F/Q383E/L412D 293/294 K91H/G183NE16D/E26A/D29R/D33E/K91H/A114H/F157Y/K158R/ ++G183NA256S/D272G/M302Q/Y371F/Q383E/L412D 295/296 K91H/G183N/F322A/E16D/E26A/D29R/D33E/K91H/A114H/F157Y/K158R/ ++ V409SG183N/A256S/D272G/M302Q/F322A/Y371F/Q383E/ V409S/L412D 297/298K91H/L194R E16D/E26A/D29R/D33E/K91H/A114H/F157Y/K158R/ ++L194R/A256S/D272G/M302Q/Y371F/Q383E/L412D 299/300 K91H/V409SE16D/E26A/D29R/D33E/K91H/A114H/F157Y/K158R/ ++A256S/D272G/M302Q/Y371F/Q383E/V409S/L412D 301/302 L24VE16D/L24V/E26A/D29R/D33E/A114H/F157Y/K158R/ ++A256S/D272G/M302Q/Y371F/Q383E/L412D 303/304 Q394EE16D/E26A/D29R/D33E/A114H/F157Y/K158R/A256S/ ++D272G/M302Q/Y371F/Q383E/Q394E/L412D 305/306 T227LE16D/E26A/D29R/D33E/A114H/F157Y/K158R/T227L/ ++A256S/D272G/M302Q/Y371F/Q383E/L412D ¹Levels of increased activity weredetermined relative to the reference polypeptide of SEQ ID NO: 174, anddefined as follows: “+” > 0.9; “++” > 1.1; and “+++” > 2.

Based on the results shown in Table 4-4, SEQ ID NO: 276 was chosen asthe parent sequence for the next iteration of protein optimization.Beneficial mutations identified based on the results shown in Table 4-4,and mutations to revert variant sequences to wild-type sequences wererecombined into this backbone. In addition, to generate new diversity,variants generated with saturation mutagenesis at different positionswere also constructed using SEQ ID NO: 276. The variants were assayed intriplicate for amylase activity at pH 6.8 after pre-incubation under twodifferent conditions. The first condition was a 1 hour gastric challengeat pH 2.75, and the second condition was a 1 hour intestinal challenge,as described in Example 3. The results (relative to the results for SEQID NO: 276) are provided in Table 4-5.

TABLE 4-5 Alpha-Amylase Activity (Relative to SEQ ID NO: 276)¹ AmylaseActivity Amylase Activity Amino Acid Amino Acid Post Intestinal PostGastric SEQ ID Differences Differences Challenge Challenge NO: (Relativeto (Relative to (Relative to (Relative to (nt/aa) SEQ ID NO: 276) SEQ IDNO: 18) SEQ ID NO: 276) SEQ ID NO: 276) 307/308 Q394GE16D/E26A/D29R/D33E/K91H/A114H/ ++ +++ A122R/I128W/E130D/F157Y/K158R/G183N/A256S/D272G/M302Q/F322A/ Y371F/Q383E/Q394G/L412D 309/310 Q394NE16D/E26A/D29R/D33E/K91H/A114H/ ++ +++ A122R/I128W/E130D/F157Y/K158R/G183N/A256S/D272G/M302Q/F322A/ Y371F/Q383E/Q394N/L412D 311/312 Q394SE16D/E26A/D29R/D33E/K91H/A114H/ ++ +++ A122R/I128W/E130D/F157Y/K158R/G183N/A256S/D272G/M302Q/F322A/ Y371F/Q383E/Q394S/L412D 313/314D16E/R29D/E33D/ E26A/K91H/A114H/A122R/I128W/ +++ A145G/S256A/E130D/A145G/F157Y/K158R/G183N/ Q302M D272G/F322A/Y371F/Q383E/L412D315/316 P349R E16D/E26A/D29R/D33E/K91H/A114H/ +++A122R/I128W/E130D/F157Y/K158R/ G183N/A256S/D272G/M302Q/F322A/Y371F/Q383E/P349R/L412D 317/318 D16E/A26E/R29D/D33E/K91H/A114H/A122R/E130D/ + +++ W128I/A145G/S256A/A145G/F157Y/K158R/G183N/D272G/ T309S/E383Q M302Q/T309S/F322A/Y371F/L412D319/320 A26E/R29D/R122A/ E16D/D33E/K91H/A114H/I128W/ + +++ D130E/A145G/A145G/F157Y/K158R/M302Q/T309S/ N183G/S256A/G272D/F322A/Y371F/Q383E/Q394E/L412D T309S/Q394E 321/322 A26E/R29D/A145G/E16D/D33E/K91H/A114H/A122R/I128W/ +++ S256A/G272D/E130D/A145G/F157Y/K158R/G183N/ Q302M/T309S/T309S/Y371F/Q383E/L412D/C481V A322F/C481V 323/324 G380RE16D/E26A/D29R/D33E/K91H/A114H/ +++ A122R/I128W/E130D/F157Y/K158R/G183N/A256S/D272G/M302Q/F322A/ Y371F/G380R/Q383E/L412D 325/326D130E/A145G/ E16D/E26A/D29R/D33E/K91H/A114H/ + +++ Q302M/T309SA122R/I128W/A145G/F157Y/K158R/ G183N/A256S/D272G/T309S/F322A/Y371F/Q383E/L412D 327/328 A26E/E33D/A145G/ E16D/D29R/K91H/A114H/A122R/++ ++ T309S/A322F/ I128W/E130D/A145G/F157Y/K158R/ E383QG183N/A256S/D272G/M302Q/T309S/ Y371F/L412D 329/330 A145G/Q302M/E16D/E26A/D29R/D33E/K91H/A114H/ ++ T309S A122R/I128W/E130D/A145G/F157Y/K158R/G183N/A256S/D272G/T309S/ F322A/Y371F/Q383E/L412D 331/332A26E/R29D/A145G/ E16D/D33E/K91H/A114H/A122R/ ++ Y157F/S256A/Q302M/I128W/E130D/A145G/K158R/G183N/ A322F/C481V D272G/Y371F/Q383E/L412D/C481V333/334 A26E/R29D/E33D/ E16D/K91H/A114H/A122R/I128W/ ++A145G/Y157F/N183G/ E130D/A145G/K158R/D272G/M302Q/ S256A/T309S/E383QT309S/F322A/Y371F/L412D 335/336 R29D/D130E/A145G/E16D/E26A/D33E/K91H/A114H/ ++ S256A/G272D/F371Y/A122R/I128W/A145G/F157Y/K158R/ Q394E G183N/M302Q/F322A/Q383E/Q394E/ L412337/338 A26E/A145G/N183G/ E16D/D29R/D33E/K91H/A114H/A122R/ + ++S256A/T309S/ I128W/E130D/A145G/F157Y/K158R/ E383QD272G/M302Q/T309S/F322A/ Y371F/L412D 339/340 D130E/A145G/S256A/E16D/E26A/D29R/D33E/K91H/A114H/ ++ G272D/F371YA122R/I128W/A145G/F157Y/K158R/ G183N/M302Q/F322A/Q383E/ L412D 341/342H91K/D130E/A145G/ E16D/E26A/D29R/D33E/A114H/A122R/ + ++ R158K/N183G/I128W/A145G/F157Y/A256S/D272G/ E383Q M302Q/F322A/Y371F/L412D 343/344D16E/A26E/A145G/ D29R/D33E/K91H/A114H/A122R/ ++ Q302MI128W/E130D/A145G/F157Y/K158R/ G183N/A256S/D272G/F322A/Y37IF/Q383E/L412D 345/346 D16E/A26E/E33D/ D29R/K91H/A114H/A122R/I128W/ ++A145G/R158K/E383Q/ E130D/A145G/F157Y/G183N/A256S/ Q394ED272G/M302Q/F322A/Y371F/Q394E/ L412D 347/348 D16E/A26E/E33D/D29R/K91H/A114H/A122R/A145G/ ++ W128I/D130E/A145G/F157Y/K158R/G183N/A256S/D272G/ Q302M/T309S/ T309S/Q383E/L412DA322F/F371Y 349/350 A26E/R29D/E33D/ E16D//K91H/A114H/A122R/I128W/ + ++D130E/A145G/Y157F/ A145G/A256S/D272G/M302Q/F322A/ R158K/N183G/Q394EY371F/Q383E/Q394E/L412D 351/352 R29D/A145G/N183G/E16D/E26A/D33E/K91H/A114H/ ++ Q302M/T309S/D412LA122R/I128W/E130D/A145G/F157Y/ K158R/A256S/D272G/T309S/F322A/Y371F/Q383E 353/354 R29D/R122A/A145G/ E16D/E26A/D33E/K91H/A114H/ ++R158K/E383Q I128W/E130D/A145G/F157Y/G183N/A256S/D272G/M302Q/F322A/Y371F/ L412D 355/356 A26E/E33D/H91K/E16D/D29R/A114H/A122R/I128W/ ++ A145G/G272D/Q302M/E130D/A145G/F157Y/K158R/G183N/ C481V A256S/F322A/Y371F/Q383E/L412D/C481V 357/358 E33D/R122A/A145G/ E16D/E26A/D29R/K91H/A114H/ ++G272D/T309S/D412L I128W/E130D/A145G/F157Y/K158R/G183N/A256S/M302Q/T309S/F322A/ Y371F/Q383E 359/360 N360GE16D/E26A/D29R/D33E/K91H/A114H/ + ++ A122R/I128W/E130D/F157Y/K158R/G183N/A256S/D272G/M302Q/F322A/ N360G/Y371F/Q383E/L412D 361/362R29D/T309S/Q394E E16D/E26A/D33E/K91H/A114H/A122R/ ++I128W/E130D/F157Y/K158R/G183N/ A256S/D272G/M302Q/T309S/F322A/Y371F/Q383E/Q394E/L412D 363/364 T309A E16D/E26A/D29R/D33E/K91H/A114H/ +++ A122R/I128W/E130D/F157Y/K158R/ G183N/A256S/D272G/M302Q/T309A/F322A/Y371F/Q383E/L412D 365/366 D16E/A26E/E33D/D29R/K91H/A122R/I128W/E130D/ + ++ H114A/A145G/T309S/A145G/F157Y/K158R/G183N/A256S/ E383Q/Q394ED272G/M302Q/T309S/F322A/Y371F/ Q394E/L412D 367/368 A26E/H114A/A145G/E16D/D29R/D33E/K91H/A122R/ + ++ A322F 1128W/E130D/A145G/F157Y/K158R/G183N/A256S/D272G/M302Q/Y371F/ Q383E/L412D 369/370 D130E/G272D/C481VE16D/E26A/D29R/D33E/K91H/A114H/ + A122R/I128W/F157Y/K158R/G183N/A256S/M302Q/F322A/Y371F/Q383E/ L412D/C481V 371/372 G463ME16D/E26A/D29R/D33E/K91H/A114H/ ++ + A122R/I128W/E130D/F157Y/K158R/G183N/A256S/D272G/M302Q/F322A/ Y371F/Q383E/L412D/G463M 373/374 S268TE16D/E26A/D29R/D33E/K91H/A114H/ + A122R/I128W/E130D/F157Y/K158R/G183N/A256S/S268T/D272G/M302Q/ F322A/Y371F/Q383E/L412D 375/376 G483TE16D/E26A/D29R/D33E/K91H/A114H/ + A122R/I128W/E130D/F157Y/K158R/G183N/A256S/D272G/M302Q/F322A/ Y371F/Q383E/L412D/G483T 377/378 G18SE16D/G18S/E26A/D29R/D33E/K91H/ ++ A114H/A122R/I128W/E130D/F157Y/K158R/G183N/A256S/D272G/M302Q/ F322A/Y371F/Q383E/L412D/E16D/ 379/380H91K/T309S/E383Q E26A/D29R/D33E/A114H/A122R/ ++I128W/E130D/F157Y/K158R/G183N/ A256S/D272G/M302Q/T309S/F322A/Y371F/L412D ¹Levels of increased activity were determined relative tothe reference polypeptide of SEQ ID NO: 276, and defined as follows:“+” > 0.9; “++” > 1.1; and “+++” > 2.

Based on the results shown in Table 4-5, SEQ ID NO: 308 was chosen asthe parent sequence for the next iteration of protein optimization.Beneficial mutations identified from Table 4-5, and diversity fromhomologs were recombined into this backbone. In addition, to generatenew diversity, variants with a single mutation at different positionswere also constructed using SEQ ID NO: 308. The variants were assayed intriplicate for amylase activity at pH 6.8 after pre-incubations undertwo different conditions. The first condition was a 2 hour gastricchallenge at pH 2.5 and the second condition was a 2 hour intestinalchallenge, as described in Example 3. The results (relative to theresults for SEQ ID NO: 308) are provided in Table 4-6.

TABLE 4.6 Alpha-Amylase Activity (Relative to SEQ ID NO: 308)¹ AmylaseActivity Amylase Activity Amino Acid Amino Acid Post Gastric PostIntestinal SEQ ID Differences Differences Challenge Challenge NO:(Relative to (Relative to (Relative to (Relative to (nt/aa) SEQ ID NO:308) SEQ ID NO: 18) SEQ ID NO: 308) SEQ ID NO: 308) 381/382 A81E/T313D/E16D/E26A/D29R/D33E/A81E/K91H/A114H/ + ++ I495LA122R/I128W/E130D/F157Y/K158R/G183N/ A256S/D272G/M302Q/T313D/F322A/Y371F/Q383E/Q394G/L412D/I495L 383/384 A81E/P259DE16D/E26A/D29R/D33E/A81E/K91H/A114H/ + +A122R/I128W/E130D/F157Y/K158R/G183N/ A256S/P259D/D272G/M302Q/F322A/Y371F/Q383E/Q394G/L412D 385/386 T313DE16D/E26A/D29R/D33E/K91H/A114H/A122R/ + + I128W/E130D/F157Y/K158R/G183N/A256S/D272G/M302Q/T313D/F322A/Y371F/ Q383E/Q394G/L412D 387/388 R29AE16D/E26A/D29A/D33E/K91H/A114H/A122R/ + + I128W/E130D/F157Y/K158R/G183N/A256S/D272G/M302Q/F322A/Y371F/Q383E/ Q394G/L412D 389/390 E473DE16D/E26A/D29R/D33E/K91H/A114H/A122R/ ++ +I128W/E130D/F157Y/K158R/G183N/ A256S/D272G/M302Q/F322A/Y371F/Q383E/Q394G/L412D/E473D 391/392 P259D/T313D/E16D/E26A/D29R/D33E/K91H/A114H/A122R/ ++ + A338SI128W/E130D/F157Y/K158R/G183N/ A256S/P259D/D272G/M302Q/T313D/F322A/A338S/Y371F/Q383E/Q394G/L412D 393/394 R92E/P257E/E16D/E26A/D29R/D33E/K91H/R92E/A114H/ ++ + K258Q/A338S/A122R/I128W/E130D/F157Y/K158R/G183N/ R468TA256S/P257E/K258Q/D272G/M302Q/ F322A/A338S/Y371F/Q383E/Q394G/L412D/R468T 395/396 F413Y E16D/E26A/D29R/D33E/K91H/A114H/A122R/ ++ +I128W/E130D/F157Y/K158R/G183N/ A256S/D272G/M302Q/F322A/Y371F/Q383E/Q394G/L412D/F413Y 397/398 K246R E16D/E26A/D29R/D33E/K91H/A114H/A122R/++ + I128W/E130D/F157Y/K158R/G183N/ K246R/A256S/D272G/M302Q/F322A/Y371F/Q383E/Q394G/L412D 399/400 M294L E16D/E26A/D29R/D33E/K91H/A114H/A122R/++ + I128W/E130D/F157Y/K158R/G183N/ A256S/D272G/M294L/M302Q/F322A/Y371F/Q383E/Q394G/L412D 401/402 E225D E16D/E26A/D29R/D33E/K91H/A114H/A122R/++ + I128W/E130D/F157Y/K158R/G183N/ E225D/A256S/D272G/M302Q/F322A/Y371F/Q383E/Q394G/L412D 403/404 F413Y/R468IE16D/E26A/D29R/D33E/K91H/A114H/A122R/ ++ +I128W/E130D/F157Y/K158R/G183N/ A256S/D272G/M302Q/F322A/Y371F/Q383E/Q394G/L412D/F413Y/R468I 405/406 R92E/K258QE16D/E26A/D29R/D33E/K91H/R92E/A114H/ ++ +A122R/I128W/E130D/F157Y/K158R/G183N/ A256S/K258Q/D272G/M302Q/F322A/Y371F/Q383E/Q394G/L412D 407/408 H448T E16D/E26A/D29R/D33E/K91H/A114H/++ + A122R/I128W/E130D/F157Y/K158R/G183N/A256S/D272G/M302Q/F322A/Y371F/Q383E/ Q394G/L412D/H448T 409/410 Q226TE16D/E26A/D29R/D33E/K91H/A114H/ ++ +A122R/I128W/E130D/F157Y/K158R/G183N/Q226T/A256S/D272G/M302Q/F322A/Y371F/ Q383E/Q394G/L412D 411/412 P349RE16D/E26A/D29R/D33E/K91H/A114H/A122R/ ++ I128W/E130D/F157Y/K158R/G183N/A256S/D272G/M302Q/F322A/P349R/Y371F/ Q383E/Q394G/L412D 413/414 V178IE16D/E26A/D29R/D33E/K91H/A114H/A122R/ ++ +I128W/E130D/F157Y/K158R/V178I/G183N/ A256S/D272G/M302Q/F322A/Y371F/Q383E/Q394G/L412D 415/416 D94G/P349R/E16D/E26A/D29R/D33E/K91H/D94G/A114H/ ++ I491LA122R/I128W/E130D/F157Y/K158R/G183N/ A256S/D272G/M302Q/F322A/P349R/Y371F/Q383E/Q394G/L412D/I49IL 417/418 E270D/P349RE16D/E26A/D29R/D33E/K91H/A114H/A122R/ ++ I128W/E130D/F157Y/K158R/G183N/A256S/E270D/D272G/M302Q/F322A/P349R/ Y371F/Q383E/Q394G/L412D 419/420D94G/E270D/ E16D/E26A/D29R/D33E/K91H/D94G/A114H/ ++ + R301K/P349R/A122R/I128W/E130D/F157Y/K158R/ G394E/D442EG183N/A256S/E270D/D272G/R301K/M302Q/F322A/P349R/Y371F/Q383E/Q394E/L412D/ D442E 421/422 A145G/E270DE16D/E26A/D29R/D33E/K91H/A114H/A122R/ ++ +I128W/E130D/A145G/F157Y/K158R/ G183N/A256S/E270D/D272G/M302Q/F322A/Y371F/Q383E/Q394G/L412D 423/424 N37T/D124N/E16D/E26A/D29R/D33E/N37T/K91H/A114H/ ++ E250D/E270D/A122R/D124N/I128W/E130D/F157Y/K158R/ P349R/G380R/G183N/E250D/A256S/E270D/D272G/ G394E/D442E/M302Q/F322A/P349R/Y371F/G380R/Q383E/ I491L Q394E/L412D/D442E/I491L425/426 E250D/P349R/ E16D/E26A/D29R/D33E/K91H/A114H/A122R/ ++ D442EI128W/E130D/F157Y/K158R/G183N/ E250D/A256S/D272G/M302Q/F322A/P349R/Y371F/Q383E/Q394G/L412D/D442E 427/428 N37T/A145GE16D/E26A/D29R/D33E/N37T/K91H/A114H/ ++ +A122R/I128W/E130D/A145G/F157Y/K158R/ G183N/A256S/D272G/M302Q/F322A/Y371F/Q383E/Q394G/L412D 429/430 N37T/A145G/E16D/E26A/D29R/D33E/N37T/K91H/A114H/ ++ + K258S/D442E/A122R/I128W/E130D/A145G/F157Y/K158R/ I491LG183N/A256S/K258S/D272G/M302Q/ F322A/Y371F/Q383E/Q394G/L412D/D442E/I491L 431/432 P349R/G380R/ E16D/E26A/D29R/D33E/K91H/A114H/ +++ D442EA122R/I128W/E130D/F157Y/K158R/G183N/A256S/D272G/M302Q/F322A/P349R/Y371F/ G380R/Q383E/Q394G/L412D/D442E433/434 D94G/Q127E/ E16D/E26A/D29R/D33E/K91H/D94G/A114H/ +++I132V/A145G/ A122R/Q127E/I128W/E130D/1132V/A145G// E250D/P349RF157Y/K158R/G183N/E250D/A256S/ D272G/M302Q/F322A/P349R/Y371F/Q383E/Q394G/L412D 435/436 M286T/A338S E16D/E26A/D29R/D33E/K91H/A114H/A122R/+++ + I128W/E130D/F157Y/K158R/G183N/A256S/D272G/M286T/M302Q/F322A/A338S/ Y371F/Q383E/Q394G/L412D 437/438M38I/P349R/ E16D/E26A/D29R/D33E/M38I/K91H/A114H/ +++ 1491LA122R/I128W/E130D/F157Y/K158R/G183N/ A256S/D272G/M302Q/F322A/P349R/Y371F/Q383E/Q394G/L412D/I491L 439/440 N37T/A145G/E16D/E26A/D29R/D33E/N37T/K91H/A114H/ +++ E250D/R301K/A122R/I128W/E130D/A145G/F157Y/K158R/ P349R/G394EG183N/E250D/A256S/D272G/R301K/ M302Q/F322A/P349R/Y371F/Q383E/Q394E/L412D ¹Levels of increased activity were determined relative to thereference polypeptide of SEQ ID NO: 308, and defined as follows: “+” >0.9; “++” > 1.1; and “+++” > 2.

Based on the results shown in Table 4-6, SEQ ID NO: 422 was chosen asthe parent sequence for the next iteration of protein optimization.Beneficial mutations identified based on the results shown in Table 4-6were recombined into this backbone. The variants were assayed insinglicate for amylase activity at pH 6.8 after pre-incubation under twodifferent conditions, the first being a 2 hour gastric challenge at pH2.3, and the second being a 2 hour intestinal challenge, as described inExample 3. The results (relative to the results for SEQ ID NO: 422) areprovided in Table 4-7.

TABLE 4-7 Alpha-Amylase Activity (Relative to SEQ ID NO: 422)¹ AmylaseActivity Amylase Activity Amino Acid Amino Acid Post Intestinal PostGastric SEQ ID Differences Differences Challenge Challenge NO: (Relativeto (Relative to (Relative to (Relative to (nt/aa) SEQ ID NO: 422) SEQ IDNO: 18) SEQ ID NO: 422) SEQ ID NO: 422) 441/442 A338S/H448TE16D/E26A/D29R/D33E/K91H/A114H/ + + A122R/I128W/E130D/A145G/F157Y/K158R/G183N/A256S/E270D/D272G/ M302Q/F322A/A338S/Y371F/Q383E/Q394G/L412D/H448T 443/444 E184K/K246R/ E16D/E26A/D29R/D33E/K91H/A114H/++ K258S/G380R A122R/I128W/E130D/A145G/F157Y/K158R/G183N/E184K/K246R/A256S/K258S/ E270D/D272G/M302Q/F322A/Y371F/G380R/Q383E/Q394G/L412D 445/446 F413Y E16D/E26A/D29R/D33E/K91H/A114H/++ + A122R/I128W/E130D/A145G/F157Y/K158R/ G183N/A256S/E270D/D272G/M302Q/F322A/Y371F/Q383E/Q394G/L412D/ F413Y 447/448 K246RE16D/E26A/D29R/D33E/K91H/A114H/ + ++A122R/I128W/E130D/A145G/F157Y/K158R/ G183N/K246R/A256S/E270D/D272G/M302Q/F322A/Y371F/Q383E/Q394G/ L412D 449/450 K246R/A338SE16D/E26A/D29R/D33E/K91H/A114H/ ++ A122R/I128W/E130D/A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/ M302Q/F322A/A338S/Y371F/Q383E/Q394G/L412D 451/452 K246R/F413Y E16D/E26A/D29R/D33E/K91H/A114H/ ++A122R/I128W/E130D/A145G/F157Y/K158R/ G183N/K246R/A256S/E270D/D272G/M302Q/F322A/Y371F/Q383E/Q394G/ L412D/F413Y 453/454 K246R/M286TE16D/E26A/D29R/D33E/K91H/A114H/ +++ ++A122R/I128W/E130D/A145G/F157Y/K158R/ G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/Q383E/ Q394G/L412D 455/456 K246R/M286T/E16D/E26A/D29R/D33E/K91H/A114H/ + ++ G380R/F413YA122R/I128W/E130D/A145G/F157Y/K158R/ G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/ Q383E/Q394G/L412D/F413Y 457/458 K246TE16D/E26A/D29R/D33E/K91H/A114H/ + A122R/I128W/E130D/A145G/F157Y/K158R/G183N/K246T/A256S/E270D/D272G/ M302Q/F322A/Y371F/Q383E/Q394G/ L412D459/460 K258S/M286T/ E16D/E26A/D29R/D33E/K91H/A114H/ ++ ++ F413YA122R/I128W/E130D/A145G/F157Y/K158R/ G183N/A256S/K258S/E270D/D272G/M286T/M302Q/F322A/Y371F/Q383E/ Q394G/L412D/F413Y 461/462 M286TE16D/E26A/D29R/D33E/K91H/A114H/ ++ ++A122R/I128W/E130D/A145G/F157Y/K158R/ G183N/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/Q383E/Q394G/ L412D 463/464 M286T/F413Y/E16D/E26A/D29R/D33E/K91H/A114H/ ++ ++ H448TA122R/I128W/E130D/A145G/F157Y/K158R/ G183N/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/Q383E/Q394G/ L412D/F413 Y/H448T 465/466 M38IE16D/E26A/D29R/D33E/M38I/K91H/ ++ ++ A114H/A122R/I128W/E130D/A145G/F157Y/K158R/G183N/A256S/E270D/ D272G/M302Q/F322A/Y371F/Q383E/Q394G/L412D 467/468 M38I/H448T E16D/E26A/D29R/D33E/M38I/K91H/ ++ ++A114H/A122R/I128W/E130D/A145G/ F157Y/K158R/G183N/A256S/E270D/D272G/M302Q/F322A/Y371F/Q383E/ Q394G/L412D/H448T 469/470 M38I/K246R/E16D/E26A/D29R/D33E/M38I/K91H/ ++ +++ M286TA114H/A122R/I128W/E130D/A145G/ F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/ Y371F/Q383E/Q394G/L412D 471/472M38I/K246R/ E16D/E26A/D29R/D33E/M38I/K91H/ + +++ M286T/G380RA114H/A122R/I128W/E130D/A145G/ F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/ Y371F/G380R/Q383E/Q394G/L412D 473/474M38I/K258S E16D/E26A/D29R/D33E/M38I/K91H/ ++ ++A114H/A122R/I128W/E130D/A145G/ F157Y/K158R/G183N/A256S/K258S/E270D/D272G/M302Q/F322A/Y371F/ Q383E/Q394G/L412D 475/476 M38I/M286TE16D/E26A/D29R/D33E/M38I/K91H/ ++ +++ A114H/A122R/I128W/E130D/A145G/F157Y/K158R/G183N/A256S/E270D/ D272G/M286T/M302Q/F322A/Y371F/Q383E/Q394G/L412D 477/478 M38I/M286T/ E16D/E26A/D29R/D33E/M38I/K91H/ +++++ F413Y/H448T A114H/A122R/I128W/E130D/A145G/F157Y/K158R/G183N/A256S/E270D/ D272G/M286T/M302Q/F322A/Y371F/Q383E/Q394G/L412D/F413Υ/H448T 479/480 M38I/M286T/E16D/E26A/D29R/D33E/M38I/K91H/ ++ ++ H448TA114H/A122R/I128W/E130D/A145G/ F157Y/K158R/G183N/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/ Q383E/Q394G/L412D/H448T 481/482M38I/Q226T/ E16D/E26A/D29R/D33E/M38I/K91H/ +++ A338S/P349R/A114H/A122R/I128W/E130D/A145G/ G380RF157Y/K158R/G183N/Q226T/A256S/E270D/D272G/M302Q/F322A/A338S/P349R/Y371F/ G380R/Q383E/Q394G/L412D 483/484M38I/Q226T/ E16D/E26A/D29R/D33E/M38I/K91H/ ++ ++ F413Y/H448TA114H/A122R/I128W/E130D/A145G/ F157Y/K158R/G183N/Q226T/A256S/E270D/D272G/M302Q/F322A/Y371F/Q383E/ Q394G/L412D/F413Y/H448T 485/486M38I/Q226T/ E16D/E26A/D29R/D33E/M38I/K91H/ + ++ K246R/K258S/A114H/A122R/I128W/E130D/A145G/ M286TF157Y/K158R/G183N/Q226T/K246R/A256S/ K258S/E270D/D272G/M286T/M302Q/F322A/Y371F/Q383E/Q394G/L412D 487/488 M38I/Q226T/E16D/E26A/D29R/D33E/M38I/K91H/ +++ K258S/M286T/A114H/A122R/I128W/E130D/A145G/ A338S/G380R/F157Y/K158R/G183N/Q226T/A256S/K258S/ H448TE270D/D272G/M286T/M302Q/F322A/ A338S/Y371F/G380R/Q383E/Q394G/L412D/H448T 489/490 M38I/Q226T/ E16D/E26A/D29R/D33E/M38I/K91H/ ++K258S/P349R/ A114H/A122R/I128W/E130D/A145G/ F413YF157Y/K158R/G183N/Q226T/A256S/ K258S/E270D/D272G/M302Q/F322A/P349R/Y371F/Q383E/Q394G/L412D/F413Y 491/492 M38I/Q226T/E16D/E26A/D29R/D33E/M38I/K91H/ ++ ++ M286TA114H/A122R/I128W/E130D/A145G/ F157Y/K158R/G183N/Q226T/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/ Q383E/Q394G/L412D 493/494M38I/Q226T/ E16D/E26A/D29R/D33E/M38I/K91H/ +++ M286T/A338S/A114H/A122R/I128W/E130D/A145G/ G380RF157Y/K158R/G183N/Q226T/A256S/E270D/ D272G/M286T/M302Q/F322A/A338S/Y371F/G380R/Q383E/Q394G/L412D 495/496 M38I/V178I/E16D/E26A/D29R/D33E/M38I/K91H/ +++ M286T/H448TA114H/A122R/I128W/E130D/A145G/ F157Y/K158R/V178I/G183N/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/ Q383E/Q394G/L412D/H448T 497/498M38I/V178I/ E16D/E26A/D29R/D33E/M38I/K91H/ +++ P349R/H448TA114H/A122R/I128W/E130D/A145G/ F157Y/K158R/V178I/G183N/A256S/E270D/D272G/M302Q/F322A/P349R/Y371F/ Q383E/Q394G/L412D/H448T 499/500M38I/V178I/ E16D/E26A/D29R/D33E/M38I/K91H/ +++ Q226T/M286T/A114H/A122R/I128W/E130D/A145G/ A338SF157Y/K158R/V178I/G183N/Q226T/A256S/ E270D/D272G/M286T/M302Q/F322A/A338S/Y371F/Q383E/Q394G/L412D 501/502 P349RE16D/E26A/D29R/D33E/K91H/A114H/ ++ A122R/I128W/E130D/A145G/F157Y/K158R/G183N/A256S/E270D/D272G/ M302Q/F322A/P349R/Y371F/Q383E/Q394G/L412D 503/504 Q226T E16D/E26A/D29R/D33E/K91H/A114H/ ++ ++A122R/I128W/E130D/A145G/F157Y/ K158R/G183N/Q226T/A256S/E270D/D272G/M302Q/F322A/Y371F/Q383E/ Q394G/L412D 505/506 Q226T/K246R/E16D/E26A/D29R/D33E/K91H/A114H/ ++ ++ F413YA122R/I128W/E130D/A145G/F157Y/ K158R/G183N/Q226T/K246R/A256S/E270D/D272G/M302Q/F322A/Y371F/Q383E/ Q394G/L412D/F413Y 507/508 Q226T/K246R/E16D/E26A/D29R/D33E/K91H/A114H/ +++ M286T/P349R/A122R/I128W/E130D/A145G/F157Y/ H448TK158R/G183N/Q226T/K246R/A256S/E270D/ D272G/M286T/M302Q/F322A/P349R/Y371F/Q383E/Q394G/L412D/H448T 509/510 Q226T/K258S/E16D/E26A/D29R/D33E/K91H/A114H/ ++ ++ M286T/F413Y/A122R/I128W/E130D/A145G/F157Y/ H448TK158R/G183N/Q226T/A256S/K258S/E270D/ D272G/M286T/M302Q/F322A/Y371F/Q383E/Q394G/L412D/F413Y/H448T 511/512 Q226T/K258S/E16D/E26A/D29R/D33E/K91H/A114H/ +++ M286T/P349R/A122R/I128W/E130D/A145G/F157Y/ G380RK158R/G183N/Q226T/A256S/K258S/E270D/ D272G/M286T/M302Q/F322A/P349R/Y371F/G380R/Q383E/Q394G/L412D 513/514 Q226T/M286TE16D/E26A/D29R/D33E/K91H/A114H/ ++ ++ A122R/I128W/E130D/A145G/F157Y/K158R/G183N/Q226T/A256S/E270D/D272G/ M286T/M302Q/F322A/Y371F/Q383E/Q394G/L412D 515/516 Q226T/M286T/ E16D/E26A/D29R/D33E/K91H/A114H/ +++G380R/H448T A122R/I128W/E130D/A145G/F157Y/K158R/G183N/Q226T/A256S/E270D/D272G/ M286T/M302Q/F322A/Y371F/G380R/Q383E/Q394G/L412D/H448T 517/518 Q226T/P349R/E16D/E26A/D29R/D33E/K91H/A114H/ ++ G380R A122R/I128W/E130D/A145G/F157Y/K158R/G183N/Q226T/A256S/E270D/D272G/ M302Q/F322A/P349R/Y371F/G380R/Q383E/Q394G/L412D 519/520 V178I E16D/E26A/D29R/D33E/K91H/A114H/ ++A122R/I128W/E130D/A145G/F157Y/ K158R/V178I/G183N/A256S/E270D/D272G/M302Q/F322A/Y371F/Q383E/Q394G/ L412D 521/522 V178I/F413YE16D/E26A/D29R/D33E/K91H/A114H/ ++ A122R/I128W/E130D/A145G/F157Y/K158R/V178I/G183N/A256S/E270D/D272G/ M302Q/F322A/Y371F/Q383E/Q394G/L412D/F413Y 523/524 V178I/G380R E16D/E26A/D29R/D33E/K91H/A114H/ ++A122R/I128W/E130D/A145G/F157Y/ K158R/V178I/G183N/A256S/E270D/D272G/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D 525/526 V178I/H448TE16D/E26A/D29R/D33E/K91H/A114H/ ++ A122R/I128W/E130D/A145G/F157Y/K158R/V178I/G183N/A256S/E270D/D272G/ M302Q/F322A/Y371F/Q383E/Q394G/L412D/H448T 527/528 V178I/K246R/ E16D/E26A/D29R/D33E/K91H/A114H/ ++A338S A122R/I128W/E130D/A145G/F157Y/K158R/V178I/G183N/K246R/A256S/E270D/ D272G/M302Q/F322A/A338S/Y371F/Q383E/Q394G/L412D 529/530 V178I/K258S/ E16D/E26A/D29R/D33E/K91H/A114H/ +++ M286T/F413Y A122R/I128W/E130D/A145G/F157Y/K158R/V178I/G183N/A256S/K258S/E270D/ D272G/M286T/M302Q/F322A/Y371F/Q383E/Q394G/L412D/F413Y 531/532 V178I/M286TE16D/E26A/D29R/D33E/K91H/A114H/ ++ A122R/I128W/E130D/A145G/F157Y/K158R/V178I/G183N/A256S/E270D/D272G/ M286T/M302Q/F322A/Y371F/Q383E/Q394G/L412D 533/534 V178I/M286T/ E16D/E26A/D29R/D33E/K91H/A114H/ +++A338S/H448T A122R/I128W/E130D/A145G/F157Y/K158R/V178I/G183N/A256S/E270D/D272G/ M286T/M302Q/F322A/A338S/Y371F/Q383E/Q394G/L412D/H448T 535/536 V178I/M286T/E16D/E26A/D29R/D33E//K91H/A114H/ ++ A338S/P349RA122R/I128W/E130D/A145G/F157Y/ K158R/V178I/G183N/A256S/E270D/D272G/M286T/M302Q/F322A/A338S/P349R/ Y371F/Q383E/Q394G/L412D 537/538V178I/Q226T/ E16D/E26A/D29R/D33E/K91H/A114H/ +++ K258S/M286T/A122R/I128W/E130D/A145G/F157Y/ Q310R/A338SK158R/V178I/G183N/Q226T/A256S/K258S/ E270D/D272G/M286T/M302Q/Q310R/F322A/A338S/Y371F/Q383E/Q394G/L412D 539/540 V178I/Q226T/E16D/E26A/D29R/D33E/K91H/A114H/ +++ M286T/A338SA122R/I128W/E130D/A145G/F157Y/ K158R/V178I/G183N/Q226T/A256S/E270D/D272G/M286T/M302Q/F322A/A338S/ Y371F/Q383E/Q394G/L412D ¹Levels ofincreased activity were determined relative to the reference polypeptideof SEQ ID NO: 422, and defined as follows: “+” > 0.9; “++” > 1.1; and“+++” > 1.5.

Based on the results from Table 4-7, SEQ ID NO: 456 was chosen as theparent sequence for the next iteration of protein optimization. Togenerate new diversity, mutations from homologs were recombined usingSEQ ID NO: 456, and variants with saturation mutagenesis at differentpositions were also constructed using this backbone. The variants wereassayed in triplicate for amylase activity under unchallenged conditionsat pH 6 and pH 7, and also at pH 6 after pre-incubation under twodifferent conditions. The first condition was a 2 hour gastric challengeat pH 2.3, and the second condition was a 2 hour intestinal challenge,as described in Example 3. The results (relative to the results for SEQID NO: 456) are provided in Table 4-8. In some additional experiments,some variants were assayed in singlicate for amylase activity at pH 6after a 2 hour gastric challenge at pH 2.3, as described in Example 3.The results (relative to the results for SEQ ID NO: 456) are provided inTable 4-9.

TABLE 4-8 Alpha-Amylase Activity (Relative to SEQ ID NO: 456)¹ AminoAcid Amino Acid Activity Post Activity Post Differences DifferencesActivity at pH 6 Activity at pH 7 Intestinal Challenge Gastric ChallengeSEQ ID NO: (Relative to (Relative to (Relative to (Relative to (Relativeto (Relative to (nt/aa) SEQ ID NO: 456) SEQ ID NO: 18) SEQ ID NO: 456)SEQ ID NO: 456) SEQ ID NO: 456) SEQ ID NO: 456) 541/542 E134RE16D/E26A/D29R/D33E/K91H/ + ++ +++ A114H/A122R/I128W/E130D/E134R/A145G/F157Y/K158R/ G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/ Y371F/G380R/Q383E/Q394G/ L412D/F413Y 543/544H210P E16D/E26A/D29R/D33E/K91H/ ++ +++ A114H/A122R/I128W/E130D/A145G/F157Y/K158R/G183N/ H210P/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/ Y371F/G380R/Q383E/Q394G/ L412D/F413Y 545/546S49A/S58T/ E16D/E26A/D29R/D33E/S49A/ ++ ++ + +++ Y141F/E298HK91H/S58T/A114H/A122R/ I128W/E130D/Y141F/A145G/ F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/ E298H/M302Q/F322A/Y371F/G380R/Q383E/Q394G/L412D/ F413Y 547/548 D279L E16D/E26A/D29R/D33E/K91H/++ ++ ++ +++ A114H/A122R/I128W/E130D/ A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/ D279L/M286T/M302Q/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y 549/550 D123RE16D/E26A/D29R/D33E/K91H/ + ++ +++ A114H/A122R/D123R/I128W/E130D/A145G/F157Y/K158R/ G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/ Y371F/G380R/Q383E/Q394G/ L412D/F413Y 551/552P417W E16D/E26A/D29R/D33E/K91H/ + + + ++ A114H/A122R/I128W/E130D/A145G/F157Y/K158R/G183N/ K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/ G380R/Q383E/Q394G/L412D/ F413Y/P417W 553/554Q302H E16D/E26A/D29R/D33E/K91H/ ++ ++ + ++ A114H/A122R/I128W/E130D/A145G/F157Y/K158R/G183N/ K246R/A256S/E270D/D272G/M286T/M302H/F322A/Y371F/ G380R/Q383E/Q394G/L412D/ F413Y 555/556 E214GE16D/E26A/D29R/D33E/K91H/ + ++ + ++ A114H/A122R/I128W/E130D/A145G/F157Y/K158R/G183N/ E214G/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/ Y371F/G380R/Q383E/Q394G/ L412D/F413Y 557/558P259C E16D/E26A/D29R/D33E/K91H/ + ++ ++ ++ A114H/A122R/I128W/E130D/A145G/F157Y/K158R/G183N/ K246R/A256S/P259C/E270D/D272G/M286T/M302Q/F322A/ Y371F/G380R/Q383E/Q394G/ L412D/F413Y 559/560T126R E16D/E26A/D29R/D33E/K91H/ ++ ++ ++ A114H/A122R/T126R/I128W/E130D/A145G/F157Y/K158R/ G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/ Y371F/G380R/Q383E/Q394G/ L412D/F413Y 561/562E131Y E16D/E26A/D29R/D33E/K91H/ + + ++ A114H/A122R/I128W/E130D/E131Y/A145G/F157Y/K158R/ G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/ Y371F/G380R/Q383E/Q394G/ L412D/F413Y 563/564Q302W E16D/E26A/D29R/D33E/K91H/ ++ ++ + ++ A114H/A122R/I128W/E130D/A145G/F157Y/K158R/G183N/ K246R/A256S/E270D/D272G/M286T/M302W/F322A/Y371F/ G380R/Q383E/Q394G/L412D/ F413Y 565/566 E393RE16D/E26A/D29R/D33E/K91H/ + + ++ A114H/A122R/I128W/E130D/A145G/F157Y/K158R/G183N/ K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/ G380R/Q383E/E393R/Q394G/ L412D/F413Y 567/568D180E E16D/E26A/D29R/D33E/K91H/ + ++ ++ ++ A114H/A122R/I128W/E130D/A145G/F157Y/K158R/D180E/ G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/ Y371F/G380R/Q383E/Q394G/ L412D/F413Y 569/570E131R E16D/E26A/D29R/D33E/K91H/ ++ A114H/A122R/I128W/E130D/E131R/A145G/F157Y/K158R/ G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/ Y371F/G380R/Q383E/Q394G/ L412D/F413Y 571/572Q302R E16D/E26A/D29R/D33E/K91H/ + ++ + ++ A114H/A122R/I128W/E130D/A145G/F157Y/K158R/G183N/ K246R/A256S/E270D/D272G/M286T/M302R/F322A/Y371F/ G380R/Q383E/Q394G/L412D/ F413Y 573/574 Q127WE16D/E26A/D29R/D33E/K91H/ ++ ++ ++ A114H/A122R/Q127W/I128W/E130D/A145G/F157Y/K158R/ G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/ Y371F/G380R/Q383E/Q394G/ L412D/F413Y 575/576S49A/S58T/ E16D/E26A/D29R/D33E/S49A/ + ++ ++ ++ V103L/L194F/S58T/K91H/V103L/A114H/ N204D/E298H A122R/I128W/E130D/A145G/F157Y/K158R/G183N/L194F/ N204D/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/ F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y577/578 D415C E16D/E26A/D29R/D33E/K91H/ + + ++ A114H/A122R/I128W/E130D/A145G/F157Y/K158R/G183N/ K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/ G380R/Q383E/Q394G/L412D/ F413Y/D415C 579/580E282T E16D/E26A/D29R/D33E/K91H/ + + ++ A114H/A122R/I128W/E130D/A145G/F157Y/K158R/G183N/ K246R/A256S/E270D/D272G/E282T/M286T/M302Q/F322A/ Y371F/G380R/Q383E/Q394G/ L412D/F413Y 581/582E131H E16D/E26A/D29R/D33E/K91H/ + ++ ++ A114H/A122R/I128W/E130D/E131H/A145G/F157Y/K158R/ G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/ Y371F/G380R/Q383E/Q394G/ L412D/F413Y 583/584Q228V E16D/E26A/D29R/D33E/K91H/ ++ ++ ++ ++ A114H/A122R/I128W/E130D/A145G/F157Y/K158R/G183N/ Q228V/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/ Y371F/G380R/Q383E/Q394G/ L412D/F413Y 585/586D428T E16D/E26A/D29R/D33E/K91H/ ++ ++ ++ ++ A114H/A122R/I128W/E130D/A145G/F157Y/K158R/G183N/ K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/ G380R/Q383E/Q394G/L412D/ F413Y/D428T 587/588S49A/S58T/ E16D/E26A/D29R/D33E/S49A/ + ++ + ++ V103L/Y141F/S58T/K91H/V103L/A114H/ F197Y/H269F/ A122R/I128W/E130D/Y141F/ M294H/E298QA145G/F157Y/K158R/G183N/ F197Y/K246R/A256S/H269F/E270D/D272G/M286T/M294H/ E298Q/M302Q/F322A/Y371F/G380R/Q383E/Q394G/L412D/ F413Y 589/590 R29E/F31L/El6D/E26A/D29E/F31L/D33E/ + + ++ R92A/A220G K91H/R92A/A114H/A122R/I128W/E130D/A145G/F157Y/ K158R/G183N/A220G/K246R/A256S/E270D/D272G/M286T/ M302Q/F322A/Y371F/G380R/Q383E/Q394G/L412D/F413Y 591/592 A319S E16D/E26A/D29R/D33E/K91H/ + + + ++A114H/A122R/I128W/E130D/ A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/ M286T/M302Q/A319S/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y 593/594 Q466CE16D/E26A/D29R/D33E/K91H/ + + ++ + A114H/A122R/I128W/E130D/A145G/F157Y/K158R/G183N/ K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/ G380R/Q383E/Q394G/L412D/ F413Y/Q466C 595/596E429S E16D/E26A/D29R/D33E/K91H/ ++ + + + A114H/A122R/I128W/E130D/A145G/F157Y/K158R/G183N/ K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/ G380R/Q383E/Q394G/L412D/ F413Y/E429S 597/598R29E/V469I E16D/E26A/D29E/D33E/K91H/ ++ ++ + + A114H/A122R/I128W/E130D/A145G/F157Y/K158R/G183N/ K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/ G380R/Q383E/Q394G/L412D/ F413Y/V469I 599/600E393S E16D/E26A/D29R/D33E/K91H/ ++ ++ ++ + A114H/A122R/I128W/E130D/A145G/F157Y/K158R/G183N/ K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/ G380R/Q383E/Q394G/E393S/ L412D/F413Y 601/602V469I E16D/E26A/D29R/D33E/K91H/ ++ ++ ++ + A114H/A122R/I128W/E130D/A145G/F157Y/K158R/G183N/ K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/ G380R/Q383E/Q394G/L412D/ F413Y/V469I 603/604A220G/V469I E16D/E26A/D29R/D33E/K91H/ + ++ + A114H/A122R/I128W/E130D/A145G/F157Y/K158R/G183N/ A220G/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/ Y371F/G380R/Q383E/Q394G/ L412D/F413Y/V469I605/606 S49A/V103L E16D/E26A/D29R/D33E/S49A/ ++ ++ ++K91H/V103L/A114H/A122R/ I128W/E130D/A145G/F157Y/K158R/G183N/K246R/A256S/ E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y 607/608 V103LE16D/E26A/D29R/D33E/K91H/ ++ ++ + V103L/A114H/A122R/I128W/E130D/A145G/F157Y/K158R/ G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/ Y371F/G380R/Q383E/Q394G/ L412D/F413Y ¹Levels ofincreased activity were determined relative to the reference polypeptideof SEQ ID NO: 456, and defined as follows: “+” > 0.9; “++” > 1.1; and“+++” > 1.5.

TABLE 4-9 Alpha-Amylase Activity (Relative to SEQ ID NO: 456)¹ AmylaseActivity Amino Acid Amino Acid Post gastric SEQ Differences DifferencesChallenge ID NO: (Relative to (Relative to (Relative to (nt/aa) SEQ IDNO: 456) SEQ ID NO: 18) SEQ ID NO: 456) 541/542 E134RE16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++++E134R/A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y 543/544H210P E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ +++A145G/F157Y/K158R/G183N/H210P/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y 547/548D279L E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/D279L/M286T/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y 549/550D123R E16D/E26A/D29R/D33E/K91H/A114H/A122R/D123R/I128W/ ++++E130D/A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y 551/552P417W E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y/P417W 553/554Q302H E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ +++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302H/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y 555/556 E214GE16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ +++A145G/F157Y/K158R/G183N/E214G/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y 557/558P259C E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++A145G/F157Y/K158R/G183N/K246R/A256S/P259C/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y 559/560T126R E16D/E26A/D29R/D33E/K91H/A114H/A122R/T126R/I128W/ +++E130D/A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y 561/562E131Y E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++++E131Y/A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y 563/564Q302W E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ +++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302W/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y 565/566 E393RE16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ +++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/E393R/ Q394G/L412D/F413Y 567/568D180E E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ +++A145G/F157Y/K158R/D180E/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y 569/570E131R E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ +++E131R/A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y 571/572Q302R E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ +++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302R/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y 573/574 Q127WE16D/E26A/D29R/D33E/K91H/A114H/A122R/Q127W/1128W/ ++++E130D/A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y577/578 D415C E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y/D415C 579/580E282T E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ +++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/E282T/M286T/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y 581/582E131H E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ +++E131H/A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y 583/584Q228V E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++++A145G/F157Y/K158R/G183N/Q228V/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y 585/586D428T E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ +++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y/D428T 591/592A319S E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ +++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/A319S/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y 593/594Q466C E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ +++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y/Q466C 595/596E429S E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ +++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y/E429S 599/600E393S E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ +++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/E393S Q394G/L412D/F413Y 821/822V409R E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/Q394G/ V409R/L412D/F413Y 823/824A319L E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/A319L/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y 825/826Y385L E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/Y385L/ Q394G/L412D/F413Y 827/828E451F E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y/E451F 829/830D180Q E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++++A145G/F157Y/K158R/D180Q/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y 831/832V409W E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/Q394G/ V409W/L412D/F413Y 833/834E282R E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ +++A145G/F157Y/K158R/E282R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y 835/836D172K E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ +++A145G/F157Y/K158R/D172K/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y 837/838R221T E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ +++A145G/F157Y/K158R/G183N/R221T/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y 839/840E492G E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ +++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y/E492G 841/842V409A E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ +++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/Q394G/ V409A/L412D/F413Y 843/844Q127R E16D/E26A/D29R/D33E/K91H/A114H/A122R/Q127R/I128W/ +++E130D/A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y 845/846D279V E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ +++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/D279V/M286T/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y 847/848D415G E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ +++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y/D415G 849/850E444R E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ +++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y/E444R 851/852E393W E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ +++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/E393W Q394G/L412D/F413Y 853/854I387V E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ +++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/I387V/ Q394G/L412D/F413Y 855/856D442W E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ +++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y/D442W 857/858R140V E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ +++R140V/A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y 859/860V470L E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y/V470L 861/862Q253R E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++A145G/F157Y/K158R/G183N/K246R/Q253R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y 863/864R92G E16D/E26A/D29R/D33E/K91H/R92G/A114H/A122R/I128W/ ++E130D/A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y 865/866E467P E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y/E467P 867/868N455T E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y/N455T 869/870E214L E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++A145G/F157Y/K158R/G183N/E214L/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y 871/872E444G E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y/E444G 873/874V470G E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y/V470G 875/876V470S E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y/V470S 877/878D123G E16D/E26A/D29R/D33E/K91H/A114H/A122R/D123G/I128W/ ++E130D/A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y 879/880E492R E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y/E492R 881/882E444V E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y/E444V 883/884D442R E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y/D442R 885/886D305R E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y D305R 887/888Q127I E16D/E26A/D29R/D33E/K91H/A114H/A122R/Q127I/I128W/ ++E130D/A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y 889/890D305W E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/D305W/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y 891/892P259W E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++A145G/F157Y/K158R/G183N/K246R/A256S/P259W/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y 893/894E359G E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/E359G/Y371F/G380R/Q383E/ Q394G/L412D/F413Y 895/896D129W E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/D129W/ ++E130D/A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y897/898 D441R E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y/D441R 899/900N455R E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y/N455R 901/902E473M E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/Q394G/L412D/ F413Y/E473M 903/904T446G E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/Q394G/L412D/ F413Y/T446G 905/906D412A E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/Q394G/L412A/ F413Y 907/908 Q127VE16D/E26A/D29R/D33E/K91H/A114H/A122R/Q127V/I128W/ ++E130D/A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y 909/910E214R E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++A145G/F157Y/K158R/G183N/E214R/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y 911/912D129C E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/D129C/ ++E130D/A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y913/914 Q127S E16D/E26A/D29R/D33E/K91H/A114H/A122R/Q127S/I128W/ ++E130D/A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y 915/916E134A E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++E134A/A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y 917/918Q302V E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302V/F322A/Y371F/G380R/Q383E/Q394G/L412D/ F413Y 919/920 E467WE16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/Q394G/L412D/ F413Y/E467W 921/922N455L E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/Q394G/L412D/ F413Y/N455L 923/924N474G E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/Q394G/L412D/ F413Y/N474G 925/926D172R E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++A145G/F157Y/K158R/D172R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y 927/928R304T E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/R304T/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y 929/930R92Q E16D/E26A/D29R/D33E/K91H/R92Q/A114H/A122R/I128W/ ++E130D/A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y 931/932A322R E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322R/Y371F/G380R/Q383E/Q394G/L412D/ F413Y 933/934 E254GE16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++A145G/F157Y/K158R/G183N/K246R/E254G/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y 935/936N455W E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/Q394G/L412D/ F413Y/N455W 937/938D180P E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++A145G/F157Y/K158R/D180P/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y 939/940D441W E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y/D441W 941/942R221V E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++A145G/F157Y/K158R/G183N/R221V/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y 943/944W346G E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/W346G/Y371F/G380R/Q383E/Q394G/ L412D/F413Y 945/946I387G E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/I387G/ Q394G/L412D/F413Y 947/948H210G E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++A145G/F157Y/K158R/G183N/H210G/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y 949/950D123C E16D/E26A/D29R/D33E/K91H/A114H/A122R/D123C/I128W/ ++E130D/A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y 951/952T384M E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/T384M/Q394G/ L412D/F413Y 953/954T126Q E16D/E26A/D29R/D33E/K91H/A114H/A122R/T126Q/I128W/ ++E130D/A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y 955/956E134F E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++E134F/A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y 957/958V494C E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/Q394G/L412D/ F413Y/V494C 959/960Q302G E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302G/F322A/Y371F/G380R/Q383E/Q394G/L412D/ F413Y 961/962 P259RE16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++A145G/F157Y/K158R/G183N/K246R/A256S/P259R/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y 963/964R459W E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y/R459W 965/966D305Q E16D/E26A/D29R/D33E/K91H/A114H/A122R/I128W/E130D/ ++A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/M302Q/D305Q/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y ¹Levels ofincreased activity were determined relative to the reference polypeptideof SEQ ID NO: 456, and defined as follows: “+” > 0.9; “++” > 1.1;“+++” > 1.5; and “++++” > 2.5.

Based on the results shown in Table 4-8, SEQ ID NO: 546 was chosen asthe parent sequence for the next iteration of protein optimization.Beneficial mutations identified from Table 4-9 were recombined into thisbackbone. The variants were assayed in triplicate for amylase activityfor unchallenged activity at pH 6 and pH 7, as well as at pH 6 afterpre-incubation under two different conditions. The first condition was a2 hour gastric challenge at pH 2.3, and the second condition was a 2hour intestinal challenge, as described in Example 3. The results(relative to the results for SEQ ID NO: 546) are provided in Table 4-10.

TABLE 4-10 Alpha-Amylase Activity (Relative to SEQ ID NO: 546)¹ AmylaseAmylase Activity Post Amino Acid Amino Acid Amylase Amylase ActivityPost Gastric Challenge Differences Differences Activity at pH 6 Activityat pH 7 Intestinal Challenge at pH 2.3 SEQ ID NO: (Relative to (Relativeto (Relative to (Relative to (Relative to (Relative to (nt/aa) SEQ IDNO: 546) SEQ ID NO: 18) SEQ ID NO: 546) SEQ ID NO: 546) SEQ ID NO: 546)SEQ ID NO: 546) 609/610 T126R/H298Q/ E16D/E26A/D29R/D33E/S49A/ + + ++Q302R/E393R/ K91H/S58T/A114H/A122R/ D428T T126R/I128W/E130D/Y141F/A145G/F157Y/K158R/G183N/ K246R/A256S/E270D/D272G/M286T/E298Q/M302R/F322A/ Y371F/G380R/Q383E/E393R/Q394G/L412D/F413Y/D428T 611/612 T126R/H210P/E16D/E26A/D29R/D33E/S49A/ + + ++ Q302R/D428T K91H/S58T/A114H/A122R/T126R/I128W/E130D/Y141F/ A145G/F157Y/K158R/G183N/H210P/K246R/A256S/E270D/ D272G/M286T/E298H/M302R/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y/D428T 613/614 D129WE16D/E26A/D29R/D33E/S49A/ + + K91H/S58T/A114H/A122R/I128W/D129W/E130D/Y141F/ A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/ M286T/E298H/M302Q/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y 615/616 T126R/H298Q/E16D/E26A/D29R/D33E/S49A/ + + ++ Q302H/E393R K91H/S58T/A114H/A122R/T126R/I128W/E130D/Y141F/ A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/ M286T/E298Q/M302H/F322A/Y371F/G380R/Q383E/E393R/ Q394G/L412D/F413Y 617/618 T126R/D180E/E16D/E26A/D29R/D33E/S49A/ + + + H298Q/Q302R/ K91H/S58T/A114H/A122R/E393R T126R/I128W/E130D/Y141F/ A145G/F157Y/K158R/D180E/G183N/K246R/A256S/E270D/ D272G/M286T/E298Q/M302R/F322A/Y371F/G380R/Q383E/ E393R/Q394G/L412D/F413Y 619/620 D123R/T126R/E16D/E26A/D29R/D33E/S49A/ + ++ H210P/H298Q/ K91H/S58T/A114H/A122R/Q302W/E393R D123R/T126R/I128W/E130D/ Y141F/A145G/F157Y/K158R/G183N/H210P/K246R/A256S/ E270D/D272G/M286T/E298Q/M302W/F322A/Y371F/G380R/ Q383E/E393R/Q394G/L412D/ F413Y 621/622D123R/T126R/ E16D/E26A/D29R/D33E/S49A/ + + ++ H210P/D428TK91H/S58T/A114H/A122R/ D123R/T126R/I128W/E130D/ Y141F/A145G/F157Y/K158R/G183N/H210P/K246R/A256S/ E270D/D272G/M286T/E298H/M302Q/F322A/Y371F/G380R/ Q383E/Q394G/L412D/F413Y/ D428T 623/624 D123RE16D/E26A/D29R/D33E/S49A/ + + + K91H/S58T/A114H/A122R/D123R/I128W/E130D/Y141F/ A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/ M286T/E298H/M302Q/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y 625/626 D123R/T126R/E16D/E26A/D29R/D33E/S49A/ + ++ D428T K91H/S58T/A114H/A122R/D123R/T126R/I128W/E130D/ Y141F/A145G/F157Y/K158R/G183N/K246R/A256S/E270D/ D272G/M286T/E298H/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y/D428T 627/628 D123R/T126R/E16D/E26A/D29R/D33E/S49A/ ++ D180Q/E214G/ K91H/S58T/A114H/A122R/E393R/D428T D123R/T126R/I128W/E130D/ Y141F/A145G/F157Y/K158R/D180Q/G183N/E214G/K246R/ A256S/E270D/D272G/M286T/E298H/M302Q/F322A/Y371F/ G380R/Q383E/E393R/Q394G/ L412D/F413Y/D428T629/630 D279L E16D/E26A/D29R/D33E/S49A/ ++ K91H/S58T/A114H/A122R/I128W/E130D/Y141F/A145G/ F157Y/K158R/G183N/K246R/A256S/E270D/D272G/D279L/ M286T/E298H/M302Q/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y 631/632 T126R/D180E/E16D/E26A/D29R/D33E/S49A/ ++ H210P/E214G/ K91H/S58T/A114H/A122R/ D428TT126R/I128W/E130D/Y141F/ A145G/F157Y/K158R/D180E/G183N/H210P/E214G/K246R/ A256S/E270D/D272G/M286T/E298H/M302Q/F322A/Y371F/ G380R/Q383E/Q394G/L412D/ F413Y/D428T 633/634T126R/E214G/ E16D/E26A/D29R/D33E/S49A/ + ++ E393R K91H/S58T/A114H/A122R/T126R/I128W/E130D/Y141F/ A145G/F157Y/K158R/G183N/E214G/K246R/A256S/E270D/ D272G/M286T/E298H/M302Q/F322A/Y371F/G380R/Q383E/ E393R/Q394G/L412D/F413Y 635/636 D123R/H210P/E16D/E26A/D29R/D33E/S49A/ ++ Q302R K91H/S58T/A114H/A122R/D123R/I128W/E130D/Y141F/ A145G/F157Y/K158R/G183N/H210P/K246R/A256S/E270D/ D272G/M286T/E298H/M302R/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y 637/638 D129W/Q228V/E16D/E26A/D29R/D33E/S49A/ ++ Q253R K91H/S58T/A114H/A122R/I128W/D129W/E130D/Y141F/ A145G/F157Y/K158R/G183N/Q228V/K246R/Q253R/A256S/ E270D/D272G/M286T/E298H/M302Q/F322A/Y371F/G380R/ Q383E/Q394G/L412D/F413Y 639/640 T126R/D180Q/E16D/E26A/D29R/D33E/S49A/ ++ H210P/E214L/ K91H/S58T/A114H/A122R/H298Q/Q302R T126R/I128W/E130D/Y141F/ A145G/F157Y/K158R/D180Q/G183N/H210P/E214L/K246R/ A256S/E270D/D272G/M286T/E298Q/M302R/F322A/Y371F/ G380R/Q383E/Q394G/L412D/ F413Y 641/642Q228V/E282T/ E16D/E26A/D29R/D33E/S49A/ +++ E444R K91H/S58T/A114H/A122R/I128W/E130D/Y141F/A145G/ F157Y/K158R/G183N/Q228V/K246R/A256S/E270D/D272G/ E282T/M286T/E298H/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y/E444R 643/644 D279L/V470SE16D/E26A/D29R/D33E/S49A/ + +++ K91H/S58T/A114H/A122R/I128W/E130D/Y141F/A145G/ F157Y/K158R/G183N/K246R/A256S/E270D/D272G/D279L/ M286T/E298H/M302Q/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y/V470S 645/646 D180Q/E214LE16D/E26A/D29R/D33E/S49A/ ++ K91H/S58T/A114H/A122R/I128W/E130D/Y141F/A145G/ F157Y/K158R/D180Q/G183N/E214L/K246R/A256S/E270D/ D272G/M286T/E298H/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y 647/648 H210P/E214L/E16D/E26A/D29R/D33E/S49A/ ++ Q302R/E393R/ K91H/S58T/A114H/A122R/ D428TI128W/E130D/Y141F/A145G/ F157Y/K158R/G183N/H210P/E214L/K246R/A256S/E270D/ D272G/M286T/E298H/M302R/F322A/Y371F/G380R/Q383E/ E393R/Q394G/L412D/F413Y/ D428T 649/650E131Y/E282T E16D/E26A/D29R/D33E/S49A/ +++ K91H/S58T/A114H/A122R/I128W/E130D/E131Y/Y141F/ A145G/F157Y/K158R/G183N/K246R/A256S/E270D/D272G/ E282T/M286T/E298H/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y 651/652 D441RE16D/E26A/D29R/D33E/S49A/ ++ K91H/S58T/A114H/A122R/I128W/E130D/Y141F/A145G/ F157Y/K158R/G183N/K246R/A256S/E270D/D272G/M286T/ E298H/M302Q/F322A/Y371F/G380R/Q383E/Q394G/L412D/ F413Y/D441R 653/654 Q253R/D279V/E16D/E26A/D29R/D33E/S49A/ ++ V470S K91H/S58T/A114H/A122R/I128W/E130D/Y141F/A145G/ F157Y/K158R/G183N/K246R/Q253R/A256S/E270D/D272G/ D279V/M286T/E298H/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y/V470S 655/656 Q253R/D279L/E16D/E26A/D29R/D33E/S49A/ ++ E492G K91H/S58T/A114H/A122R/I128W/E130D/Y141F/A145G/ F157Y/K158R/G183N/K246R/Q253R/A256S/E270D/D272G/ D279L/M286T/E298H/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y/E492G 657/658 D123R/T126R/E16D/E26A/D29R/D33E/S49A/ ++ D180E/E214L/ S58T/K91H/A114H/A122R/H298Q/Q302W/ D123R/T126R/I128W/E130D/ E393R Y141F/A145G/F157Y/K158R/D180E/G183N/E214L/K246R/ A256S/E270D/D272G/M286T/E298Q/M302W/F322A/Y371F/ G380R/Q383E/Q394G/L412D/ F413Y 659/660E131H/Q228V/ E16D/E26A/D29R/D33E/S49A/ ++ D279V S58T/K91H/A114H/A122R/I128W/E130D/E131H/Y141F/ A145G/F157Y/K158R/G183N/Q228V/K246R/A256S/E270D/ D272G/D279V/M286T/E298H/M302Q/F322A/Y371F/G380R/ Q383E/Q394G/L412D/F413Y ¹Levels of increasedactivity were determined relative to the reference polypeptide of SEQ IDNO: 546, and defined as follows: “+” > 0.9; “++” > 1.1; and “+++” > 1.5.

Based on the results shown in Table 4-10, SEQ ID NO: 632 was chosen asthe parent sequence for the next iteration of protein optimization. Togenerate new diversity, mutations from homologs were recombined usingthe backbone. In addition, variants produced using saturationmutagenesis at different positions were also constructed on thisbackbone. The variants were assayed in triplicate for amylase activityfor unchallenged activity at pH 6, and also after pre-incubation undertwo different conditions. The first condition was a 2 hour gastricchallenge at pH 2.3, and the second condition was a 2 hour intestinalchallenge, as described in Example 3. The results (relative to theresults for SEQ ID NO: 632) are provided in Table 4-11.

TABLE 4-11 Alpha-Amylase Activity (Relative to SEQ ID NO: 632)¹ Amylaseactivity Amylase Activity Amino Acid Amino Acid Amylase Activity PostIntestinal Post Gastric SEQ ID Differences Differences at pH 6 ChallengeChallenge NO: (Relative to (Relative to (Relative to (Relative to SEQ(Relative to SEQ (nt/aa) SEQ ID NO: 632) SEQ ID NO: 18) SEQ ID NO: 632)ID NO: 632) ID NO: 632) 661/662 H165V E16D/E26A/D29R/D33E/S49A/S58T/ +++++ + K91H/A114H/A122R/T126R/I128W/ E130D/Y141F/A145G/F157Y/K158R/H165V/D180E/G183N/H210P/E214G/ K246R/A256S/E270D/D272G/M286T/E298H/M302Q/F322A/Y371F/G380R/ Q383E/Q394G/L412D/F413Y/D428T 663/664E184D/V223L/Y30 E16D/E26A/D29R/D33E/S49A/S58T/ ++ ++ ++ 6F/M437LK91H/A114H/A122R/T126R/I128W/ E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/E184D/H210P/E214G/ V223L/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/Y306F/F322A/ Y371F/G380R/Q383E/Q394G/L412D/F413Y/D428T/M437L 665/666 I166L/E203S E16D/E26A/D29R/D33E/S49A/S58T/ ++++ K91H/A114H/A122R/T126R/I128W/ E130D/Y141F/A145G/F157Y/K158R/I166L/D180E/G183N/E203S/H210P/ E214G/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/F322A/Y371F/ G380R/Q383E/Q394G/L412D/F413Y/D428T667/668 Y52R E16D/E26A/D29R/D33E/S49A/Y52R/S58T/ ++ ++ ++K91H/A114H/A122R/T126R/I128W/ E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/H210P/E214G/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/F322A/Y371F/ G380R/Q383E/Q394G/L412D/F413Y/D428T669/670 E184D/I216L/Y306 E16D/E26A/D29R/D33E/S49A/S58T/K91H/ ++ ++ +F/M308L A114H/A122R/T126R/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/E184D/H210P/E214G/I216L/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/Y306F/M308L/F322A/Y371F/G380R/Q383E/Q394G/L412D/ F413Y/D428T 671/672 D396EE16D/E26A/D29R/D33E/S49A/S58T/K91H/ ++ ++ ++A114H/A122R/T126R/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/H210P/E214G/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/D396E/L412D/F413Y/D428T673/674 H114R/V223L/Y30 E16D/E26A/D29R/D33E/S49A/S58T/K91H/ ++ ++ +6F/M308L A114R/A122R/T126R/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/H210P/E214G/V223L/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/Y306F/M308L/F322A/Y371F/G380R/Q383E/Q394G/L412D/F413Y/D428T 675/676 D396QE16D/E26A/D29R/D33E/S49A/S58T/K91H/ ++ ++ ++A114H/A122R/T126R/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/H210P/E214G/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/D396Q/L412D/F413Y/D428T677/678 A48D/I166L/ E16D/E26A/D29R/D33E/A48D/S49A/S58T/ ++ ++D170S/E203S K91H/A114H/A122R/T126R/1128W/E130D/Y141F/A145G/F157Y/K158R/I166L/D170S/D180E/G183N/E203S/H210P/E214G/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/F322A/Y371F/G380R/Q383E/Q394G/L412D/ F413Y/D428T 679/680 H448PE16D/E26A/D29R/D33E/S49A/S58T/K91H/ ++ + +A114H/A122R/T126R/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/H210P/E214G/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y/D428T/H448P681/682 Y45S/I166L/H298D E16D/E26A/D29R/D33E/Y45S/S49A/S58T/ ++K91H/A114H/A122R/T126R/I128W/E130D/ Y141F/A145G/F157Y/K158R/I166L/D180E/G183N/H210P/E214G/K246R/A256S/E270D/D272G/M286T/E298D/M302Q/F322A/Y371F/ G380R/Q383E/Q394G/L412D/F413Y/D428T683/684 M294A E16D/E26A/D29R/D33E/S49A/S58T/K91H/ ++ ++ ++A114H/A122R/T126R/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/H210P/E214G/K246R/A256S/E270D/D272G/M286T/M294A/E298H/M302Q/F322A/Y371F/G380R/ Q383E/Q394G/L412D/F413Y/D428T685/686 V255M E16D/E26A/D29R/D33E/S49A/S58T/K91H/ ++ + +A114H/A122R/T126R/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/H210P/E214G/K246R/V255M/A256S/E270D/D272G/M286T/E298H/M302Q/F322A/Y371F/G380R/ Q383E/Q394G/L412D/F413Y/D428T687/688 T58A/I166L E16D/E26A/D29R/D33E/S49A/S58A/K91H/ ++ +A114H/A122R/T126R/I128W/E130D/Y141F/A145G/F157Y/K158R/I166L/D180E/G183N/H210P/E214G/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/F322A/Y371F/G380R/ Q383E/Q394G/L412D/F413Y/D428T689/690 A48D E16D/E26A/D29R/D33E/A48D/S49A/S58I/ ++ ++K91H/A114H/A122R/T126R/I128W/E130D/ Y141F/A145G/F157Y/K158R/D180E/G183N/H210P/E214G/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/F322A/Y371F/G380R/ Q383E/Q394G/L412D/F413 Y/D428T691/692 Q189D E16D/E26A/D29R/D33E/S49A/S58T/K91H/ ++A114H/A122R/T126R/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/Q189D/H210P/E214G/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/F322A/Y371F/G380R/ Q383E/Q394G/L412D/F413Y/D428T693/694 H114R E16D/E26A/D29R/D33E/S49A/S58T/K91H/ ++ ++ +A114R/A122R/T126R/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/H210P/E214G/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y/D428T 695/696Q82A E16D/E26A/D29R/D33E/S49A/S58T/Q82A/ ++ + +K91H/A114H/A122R/T126R/I128W/E130D/ Y141F/A145G/F157Y/K158R/D180E/G183N/H210P/E214G/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/F322A/Y371F/G380R/ Q383E/Q394G/L412D/F413Y/D428T697/698 T58A/E203S E16D/E26A/D29R/D33E/S49A/S58A/K91H/ ++ ++A114H/A122R/T126R/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/H210P/E214G/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y/D428T 699/700N482L E16D/E26A/D29R/D33E/S49A/S58T/K91H/ ++ + +A114H/A122R/T126R/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/E203S/H210P/E214G/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/F322A/Y371F/G380R/ Q383E/Q394G/L412D/F413Y/D428T701/702 S72T E16D/E26A/D29R/D33E/S49A/S58T/S72T/ ++ ++ ++K91H/A114H/A122R/T126R/I128W/E130D/ Y141F/A145G/F157Y/K158R/D180E/G183N/H210P/E214G/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/F322A/Y371F/G380R/ Q383E/Q394G/L412D/F413Y/D428T703/704 G483A E16D/E26A/D29R/D33E/S49A/S58T/K91H/ ++ + ++A114H/A122R/T126R/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/H210P/E214G/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y/D428T/G483A705/706 E397M E16D/E26A/D29R/D33E/S49A/S58T/K91H/ ++ ++ ++A114H/A122R/T126R/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/H210P/E214G/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/E397M/L412D/F413Y/D428T707/708 I495G E16D/E26A/D29R/D33E/S49A/S58T/K91H/ ++ +A114H/A122R/T126R/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/H210P/E214G/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y/D428T/I495G709/710 K258S E16D/E26A/D29R/D33E/S49A/S58T/K91H/ + +A114H/A122R/T126R/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/H210P/E214G/K246R/A256S/K258S/E270D/D272G/M286T/E298H/M302Q/F322A/Y371F/G380R/ Q383E/Q394G/L412D/F413Y/D428T711/712 H298N E16D/E26A/D29R/D33E/S49A/S58T/K91H/ + ++ ++A114H/A122R/T126R/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/H210P/E214G/K246R/A256S/E270D/D272G/M286T/E298N/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y/D428T 713/714I495L E16D/E26A/D29R/D33E/S49A/S58T/K91H/ + + +A114H/A122R/T126R/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/H210P/E214G/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y/D428T/I495L715/716 E203S E16D/E26A/D29R/D33E/S49A/S58T/K91H/ + ++A114H/A122R/T126R/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/E203S/H210P/E214G/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/F322A/Y371F/G380R/ Q383E/Q394G/L412D/F413Y/D428T717/718 R468V E16D/E26A/D29R/D33E/S49A/S58T/K91H/ + ++ ++A114H/A122R/T126R/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/H210P/E214G/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y/D428T/R468V719/720 Q275I E16D/E26A/D29R/D33E/S49A/S58T/K91H/ + ++ +A114H/A122R/T126R/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/H210P/E214G/K246R/A256S/E270D/D272G/Q275I/M286T/E298H/M302Q/F322A/Y371F/G380R/ Q383E/Q394G/L412D/F413Y/D428T721/722 E167Y E16D/E26A/D29R/D33E/S49A/S58T/K91H/ + ++ +A114H/A122R/T126R/I128W/E130D/Y141F/A145G/F157Y/K158R/E167Y/D180E/G183N/H210P/E214G/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/F322A/Y371F/G380R/ Q383E/Q394G/L412D/F413Y/D428T723/724 Q149K E16D/E26A/D29R/D33E/S49A/S58T/K91H/ + + +A114H/A122R/T126R/I128W/E130D/Y141F/A145G/Q149K/F157Y/K158R/D180E/G183N/H210P/E214G/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/F322A/Y371F/G380R/ Q383E/Q394G/L412D/F413Y/D428T725/726 V255R E16D/E26A/D29R/D33E/S49A/S58T/K91H/ + + +A114H/A122R/T126R/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/H210P/E214G/K246R/V255R/A256S/E270D/D272G/M286T/E298H/M302Q/F322A/Y371F/G380R/ Q383E/Q394G/L412D/F413Y/D428T727/728 K284M E16D/E26A/D29R/D33E/S49A/S58T/K91H/ + ++ +A114H/A122R/T126R/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/H210P/E214G/K246R/K284M/A256S/E270D/D272G/M286T/E298H/M302Q/F322A/Y371F/G380R/ Q383E/Q394G/L412D/F413Y/D428T729/730 H298D E16D/E26A/D29R/D33E/S49A/S58T/K91H/ + + +A114H/A122R/T126R/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/H210P/E214G/K246R/A256S/E270D/D272G/M286T/E298D/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y/D428T 731/732D170S E16D/E26A/D29R/D33E/S49A/S58T/K91H/ + + +A114H/A122R/T126R/I128W/E130D/Y141F/A145G/F157Y/K158R/D170S/D180E/G183N/H210P/E214G/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/F322A/Y371F/G380R/ Q383E/Q394G/L412D/F413Y/D428T733/734 E225T E16D/E26A/D29R/D33E/S49A/S58T/K91H/ + +A114H/A122R/T126R/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/H210P/E214G/E225T/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/F322A/Y371F/G380R/ Q383E/Q394G/L412D/F413Y/D428T735/736 T411E E16D/E26A/D29R/D33E/S49A/S58T/K91H/ + + +A114H/A122R/T126R/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/H210P/E214G/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/T411E/F413Y/D428T737/738 Q280C E16D/E26A/D29R/D33E/S49A/S58T/K91H/ + ++A114H/A122R/T126R/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/H210P/E214G/K246R/A256S/E270D/D272G/Q280C/M286T/E298H/M302Q/F322A/Y371F/G380R/ Q383E/Q394G/L412D/F413Y/D428T739/740 Q275R E16D/E26A/D29R/D33E/S49A/S58T/K91H/ + ++ +A114H/A122R/T126R/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/H210P/E214G/K246R/A256S/E270D/D272G/Q275R/M286T/E298H/M302Q/F322A/Y371F/G380R/ Q383E/Q394G/L412D/F413Y/D428T741/742 R2L R2L/E16D/E26A/D29R/D33E/S49A/S58T/ + + +K91H/A114H/A122R/T126R/I128W/E130D/ Y141F/A145G/F157Y/K158R/D180E/G183N/H210P/E214G/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/F322A/Y371F/G380R/ Q383E/Q394G/L412D/F413Y/D428T743/744 K284L E16D/E26A/D29R/D33E/S49A/S58T/K91H/ + + ++A114H/A122R/T126R/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/H210P/E214G/K246R/A256S/E270D/D272G/K284L/M286T/E298H/M302Q/F322A/Y371F/G380R/ Q383E/Q394G/L412D/F413Y/D428T745/746 P431T E16D/E26A/D29R/D33E/S49A/S58T/K91H/ + +A114H/A122R/T126R/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/H210P/E214G/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y/D428T/P431T¹Levels of increased activity were determined relative to the referencepolypeptide of SEQ ID NO: 632, and defined as follows: “+” > 0.9; “++” >1.1; and “+++” > 1.5.

Based on the results shown in Table 4-11, SEQ ID NO: 664 was chosen asthe parent sequence for the next iteration of protein optimization.Mutations from homologs, and any other beneficial mutations identifiedfrom all the screening results not yet incorporated were recombined onthis backbone. In addition, variants produced using saturationmutagenesis at different positions were also constructed on thisbackbone. The variants were assayed in triplicate for amylase activityfor unchallenged activity at pH 6, and also after pre-incubation undertwo different conditions. The first condition was a 2 hour gastricchallenge at pH 2.3, and the second condition was a 2 hour intestinalchallenge, as described in Example 3. The results (relative to theresults for SEQ ID NO: 664) are provided in Table 4-12.

In additional experiments, some of the variants from Table 4-12 werealso scaled up for production in shake flasks, and the purified enzymeswere assayed for amylase activity for unchallenged activity at pH 6, andalso after pre-incubations under two different conditions. The firstcondition was a 2 hour gastric challenge at pH 3, and the secondcondition was a 3 hour intestinal challenge, as described in Example 3.The results (relative to the results for SEQ ID NO: 664) are provided inTable 4-13.

TABLE 4-12 Alpha-Amylase Activity (Relative to SEQ ID NO: 664)¹ AmylaseActivity Amylase Activity Amino Acid Amino Acid Amylase Activity PostIntestinal Post Gastric SEQ ID Differences Differences at pH 6 ChallengeChallenge NO: (Relative to (Relative to (Relative to (Relative to(Relative to (nt/aa) SEQ ID NO: 664) SEQ ID NO: 18) SEQ ID NO: 664) SEQID NO: 664) SEQ ID NO: 664) 747/748 D123E/F197Y/E22E16D/E26A/D29R/D33E/S49A/S58T/K91H/ ++ ++ ++ 5G/M294A/D396Q/A114H/A122R/D123E/T126R/I128W/E130D/ H448Q/V469IY141F/A145G/F157Y/K158R/D180E/G183N/E184D/F197Y/H210P/E214G/V223L/E225G/K246R/A256S/E270D/D272G/M286T/M294A/E298H/M302Q/Y306F/F322A/Y371F/G380R/Q383E/Q394G/D396Q/L412D/F413Y/D428T/ M437L/H448Q/V469I 749/750 H500PE16D/E26A/D29R/D33E/S49A/S58T/K91H/ ++ + ++A114H/A122R/T126R/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/E184D/H210P/E214G/V223L/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/Y306F/F322A/Y371F/G380R/Q383E/Q394G/L412D/F413Y/ D428T/M437L/H500P 751/752D123E/F197Y E16D/E26A/D29R/D33E/S49A/S58T/K91H/ ++ ++ ++A114H/A122R/D123E/T126R/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/E184D/F197Y/H210P/E214G/V223L/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/Y306F/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y/D428T/M437L 753/754I392C/H503L E16D/E26A/D29R/D33E/S49A/S58T/K91H/ ++A114H/A122R/T126R/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/E184D/H210P/E214G/V223L/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/Y306F/F322A/Y371F/G380R/Q383E/I392C/Q394G/L412D/ F413Y/D428T/M437L/H503L 755/756W458F E16D/E26A/D29R/D33E/S49A/S58T/K91H/ + +A114H/A122R/T126R/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/E184D/H210P/E214G/V223L/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/Y306F/F322A/Y371F/G380R/Q383E/Q394G/L412D/F413Y/ D428T/M437L/W458F 757/758 L61YE16D/E26A/D29R/D33E/S49A/S58T/L61Y/ + + +K91H/A114H/A122R/T126R/I128W/E130D/ Y141F/A145G/F157Y/K158R/D180E/G183N/E184D/H210P/E214G/V223L/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/Y306F/F322A/Y371F/G380R/Q383E/Q394G/L412D/ F413Y/D428T/M437L 759/760E225G/M294A/H2 E16D/E26A/D29R/D33E/S49A/S58T/K91H/ + ++ + 98N/D396QA114H/A122R/T126R/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/E184D/H210P/E214G/V223L/E225G/K246R/A256S/E270D/D272G/M286T/M294A/E298N/M302Q/Y306F/F322A/Y371F/G380R/Q383E/Q394G/ D396Q/L412D/F413Y/D428I/M437L761/762 L41F E16D/E26A/D29R/D33E/L41F/S49A/S58T/ + +K91H/A114H/A122R/T126R/I128W/E130D/ Y141F/A145G/F157Y/K158R/D180E/G183N/E184D/H210P/E214G/V223L/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/Y306F/F322A/Y371F/G380R/Q383E/Q394G/L412D/ F413Y/D428T/M437L 763/764D123E/D170S/F19 E16D/E26A/D29R/D33E/S49A/S58T/K91H/ + ++ +7Y/Q275I/H298N/ A14H/A122R/D123E/T126R/I128W/E130D/ D396Q/H448Q/G4Y141F/A145G/F157Y/K158R/D170S/D180E/ 83QG183N/E184D/F197Y/H210P/E214G/V223L/K246R/A256S/E270D/D272G/Q275I/M286T/E298N/M302Q/Y306F/F322A/Y371F/G380R/Q383E/Q394G/D396Q/L412D/F413Y/D428T/ M437L/H448Q/G483Q 765/766 W458YE16D/E26A/D29R/D33E/S49A/S58T/K91H/ + ++ +A114H/A122R/T126R/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/E184D/H210P/E214G/V223L/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/Y306F/F322A/Y371F/G380R/Q383E/Q394G/L412D/F413Y/ D428T/M437L/W458Y 767/768 G499RE16D/E26A/D29R/D33E/S49A/S58T/K91H/ + + +A114H/A122R/T126R/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/E184D/H210P/E214G/V223L/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/Y306F/F322A/Y371F/G380R/Q383E/Q394G/L412D/F413Y/ D428T/M437L/G499R 769/770D170S/Q275I/M29 E16D/E26A/D29R/D33E/S49A/S58T/K91H/ + + + 4A/H298N/H448QA114H/A122R/T126R/I128W/E130D/Y141F/A145G/F157Y/K158R/D170S/D180E/G183N/E184D/H210P/E214G/V223L/K246R/A256S/E270D/D272G/Q275I/M286T/M294A/E298N/M302Q/Y306F/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y/D428T/M437L/H448Q771/772 D123E/K284L/M2 E16D/E26A/D29R/D33E/S49A/S58T/K91H/ + ++ ++94A/A324T/D396 A114H/A122R/D123E/T126R/I128W/E130D/ Q/H448QY141F/A145G/F157Y/K158R/D180E/G183N/E184D/H210P/E214G/V223L/K246R/A256S/E270D/D272G/K284L/M286T/M294A/E298H/M302Q/Y306F/F322A/A324T/Y371F/G380R/Q383E/Q394G/D396Q/L412D/F413Y/D428T/ M437L/H448Q 773/774 H165V/E225G/K2E16D/E26A/D29R/D33E/S49A/S58T/K91H/ + ++ + 84L/H298N/V469I/A114H/A122R/T126R/I128W/E130D/Y141F/ G483QA145G/F157Y/K158R/H165V/D180E/G183N/E184D/H210P/E214G/V223L/E225G/K246R/A256S/E270D/D272G/K284L/M286T/E298N/M302Q/Y306F/F322A/Y371F/G380R/Q383E/Q394G/L412D/F413Y/D428T/M437L/V469I/ G483Q 775/776 I95V/I205VE16D/E26A/D29R/D33E/S49A/S58T/K91H/ + +I95V/A114H/A122R/T126R/I128W/E130D/ Y141F/A145G/F157Y/K158R/D180E/G183N/E184D/I205V/H210P/E214G/V223L/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/Y306F/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y/D428T/M437L 777/778T227C E16D/E26A/D29R/D33E/S49A/S58I/K91H/ + + ++A114H/A122R/T126R/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/E184D/H210P/E214G/V223L/T227C/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/Y306F/F322A/Y371F/G380R/Q383E/Q394G/L412D/ F413Y/D428T/M437L 779/780I205V/D206E E16D/E26A/D29R/D33E/S49A/S58I7K91H/ + + ++A114H/A122R/T126R/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/E184D/I205V/D206E/H210P/E214G/V223L/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/Y306F/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y/D428T/M437L 781/782T58S/H500P/ E16D/E26A/D29R/D33E/S49A/K91H/A114H/ + ++ + H501PA122R/T126R/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/E184D/H210P/E214G/V223L/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/Y306F/F322A/Y371F/G380R/Q383E/Q394G/L412D/F413Y/D428I/ M437L/H500P/H501P 783/784I205V/D206E/M22 E16D/E26A/D29R/D33E/S49A/S58T/K91H/ ++ 4L/T315S/W458F/A114H/A122R/T126R/I128W/E130D/Y141F/ F480LA145G/F157Y/K158R/D180E/G183N/E184D/I205V/D206E/H210P/E214G/V223L/M224L/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/Y306F/T315S/F322A/Y371F/G380R/Q383E/Q394G/L412D/F413Y/D428T/M437L/ W458F/F480L 785/786I95L/V252I/T285V/ E16D/E26A/D29R/D33E/S49A/S58T/K91H/ ++ A324TI95L/A114H/A122R/T126R/I128W/E130D/ Y141F/A145G/F157Y/K158R/D180E/G183N/E184D/H210P/E214G/V223L/K246R/V252I/A256S/E270D/D272G/T285V/M286T/E298H/ M302Q/Y306F/F322A/A324TY371F/G380R/Q383E/Q394G/L412D/F413Y/D428T/M437L 787/788 I95V/L98I/T227V/E16D/E26A/D29R/D33E/S49A/S58T/K91H/ + S423VI95V/L98I/A114H/A122R/T126R/I128W/ E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/E184D/H210P/E214G/V223L/T227V/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/Y306F/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y/S423V/D428T/M437L789/790 I95V/L98I/I205V/ E16D/E26A/D29R/D33E/S49A/S58T/K91H/ ++T227V/S423V/W4 I95V/L98I/A114H/A122R/T126R/I128W/ 58FE130D/Y141F/A145G/F157Y/K158R/D180E/G183N/E184D/I205V/H210P/E214G/V223L/T227V/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/Y306F/F322A/Y371F/G380R/Q383E/Q394G/L412D/F413Y/S423V/D428T/ M437L/W458F 791/792 D206E/T227VE16D/E26A/D29R/D33E/S49A/S58T/K91H/ + ++A114H/A122R/T126R/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/E184D/D206E/H210P/E214G/V223L/T227V/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/Y306F/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y/D428T/M437L 793/794L6I/V39I/V103D/ L6I/E16D/E26A/D29R/D33E/V39I/S49A/ + A324T/I420VS58T/K91H/V103D/A114H/A122R/T126R/ I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/E184D/H210P/E214G/V223L/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/Y306F/F322A/A324T/Y371F/G380R/Q383E/Q394G/L412D/F413Y/I420V/D428T/ M437L 795/796 D206E/T227V/T31E16D/E26A/D29R/D33E/S49A/S58T/K91H/ ++ 5S/S423VA114H/A122R/T126R/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/E184D/D206E/H210P/E214G/V223L/T227V/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/Y306F/T315S/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y/S423V/D428T/M437L797/798 E134R/E225G/Q27 E16D/E26A/D29R/D33E/S49A/S58T/K91H/ ++ 5I/Q302WA114H/A122R/T126R/I128W/E130D/E134R/Y141F/A145G/F157Y/K158R/D180E/G183N/E184D/H210P/E214G/V223L/E225G/K246R/A256S/E270D/D272G/Q275I/M286T/E298H/M302W/Y306F/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y/D428T/M437L¹Levels of increased activity were determined relative to the referencepolypeptide of SEQ ID NO: 664, and defined as follows: “+” > 0.9; and“++” > 1.1.

TABLE 4-13 Alpha-Amylase Activity (Relative to SEQ ID NO: 664)¹ AmylaseActivity Amylase Activity Amino Acid Amino Acid Amylase Activity PostGastric Post Intestinal SEQ ID Differences Differences at pH 6 ChallengeChallenge NO: (Relative to SEQ (Relative to (Relative to (Relative to(Relative to (nt/aa) ID NO: 664) SEQ ID NO: 18) SEQ ID NO: 664) SEQ IDNO: 664) SEQ ID NO: 664) 797/798 E134R/E225G/Q27E16D/E26A/D29R/D33E/S49A/S58T/K91H/ + ++ 5I/Q302WA114H/A122R/T126R/I128W/E130D/E134R/Y141F/A145G/F157Y/K158R/D180E/G183N/E184D/H210P/E214G/V223L/E225G/K246R/A256S/E270D/D272G/Q275I/M286T/E298H/M302W/Y306F/F322A/Y371F/G380R/Q383E/ Q394G/L412D/F413Y/D428T/M437L747/748 D123E/F197Y/E22 E16D/E26A/D29R/D33E/S49A/S58T/K91H/ + ++ +5G/M294A/D396Q/ A114H/A122R/D123E/T126R/I128W/E130D/ H448Q/V469IY141F/A145G/F157Y/K158R/D180E/G183N/E184D/F197Y/H210P/E214G/V223L/E225G/K246R/A256S/E270D/D272G/M286T/M294A/E298H/M302Q/Y306F/F322A/Y371F/G380R/Q383E/Q394G/D396Q/L412D/F413Y/D428T/ M437L/H448Q/V469I 751/752D123E/F197Y E16D/E26A/D29R/D33E/S49A/S58T/K91H/ + ++ ++A114H/A122R/D123E/T126R/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/E184D/F197Y/H210P/E214G/V223L/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/Y306F/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y/D428T/M437L 783/784I205V/D206E/M22 E16D/E26A/D29R/D33E/S49A/S58T/K91H/ + ++ 4L/T315S/W458F/A114H/A122R/T126R/I128W/E130D/Y141F/ F480LA145G/F157Y/K158R/D180E/G183N/E184D/I205V/D206E/H210P/E214G/V223L/M224L/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/Y306F/T315S/F322A/Y371F/G380R/Q383E/Q394G/L412D/F413Y/D428T/M437L/ W458F/F480L 785/786I95L/V252I/T285V/ E16D/E26A/D29R/D33E/S49A/S58T/K91H/ + + A324TI95L/A114H/A122R/T126R/I128W/E130D/ Y141F/A145G/F157Y/K158R/D180E/G183N/E184D/H210P/E214G/V223L/K246R/V252I/A256S/E270D/D272G/T285V/M286T/E298H/M302Q/Y306F/F322A/A324T/Y371F/G380R/ Q383E/Q394G/L412D/F413Y/D428T/M437L779/780 I205V/D206E E16D/E26A/D29R/D33E/S49A/S58T/K91H/ + ++A114H/A122R/T126R/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/E184D/I205V/D206E/H210P/E214G/V223L/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/Y306F/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y/D428T/M437L 663/664 —— + + + ¹Levels of increased activity were determined relative to thereference polypeptide of SEQ ID NO: 664, and defined as follows: “+” >0.9; and “++” > 1.1.

Based on the results shown in Table 4-10, SEQ ID NO: 632 was found tohave improved gastric stability, but lower intestinal stability comparedto SEQ ID NO: 546. Sequence analysis identified a T126R substitutionthat may have caused the compromised intestinal stability. Based on theresults from Table 4-13, SEQ ID NO: 780 was chosen as the parentsequence for the next iteration of protein optimization. An NNK libraryat position 126 was constructed using SEQ ID NO: 780, to rescue the lossof intestinal stability while maintaining gastric stability. Thevariants were assayed for amylase activity at pH 6 after pre-incubationunder three different conditions. The first condition was a 2 hourgastric challenge at pH 2.3, the second condition was a 2 hour trypsinonly challenge (no chymotrypsin), and the third condition was a 2 hourincubation with a protease (SEQ ID NO: 20), as described in Example 3.The results (relative to the results for SEQ ID NO: 780) are provided inTable 4-14.

TABLE 4-14 Alpha-Amylase Activity (Relative to SEQ ID NO: 780)¹ AmylaseActivity Amylase Activity Amylase Activity Amino Acid Amino Acid PostGastric Post Intestinal Post Protease SEQ ID Differences DifferencesChallenge Challenge Challenge NO: (Relative to (Relative to (Relative to(Relative to (Relative to (nt/aa) SEQ ID NO: 780) SEQ ID NO: 18) SEQ IDNO: 780) SEQ ID NO: 780) SEQ ID NO: 780) 799/800 R126NE16D/E26A/D29R/D33E/S49A/S58T/K91H/ + ++ +++A114H/A122R/T126N/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/E184D/I205V/D206E/H210P/E214G/V223L/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/Y306F/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y/D428T/M437L 801/802R126L E16D/E26A/D29R/D33E/S49A/S58T/K91H/ + + ++A114H/A122R/T126L/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/E184D/I205V/D206E/H210P/E214G/V223L/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/Y306F/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y/D428T/M437L 803/804R126Q E16D/E26A/D29R/D33E/S49A/S58T/K91H/ + ++A114H/A122R/T126Q/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/E184D/I205V/D206E/H210P/E214G/V223L/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/Y306F/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y/D428T/M437L 805/806R126K E16D/E26A/D29R/D33E/S49A/S58T/K91H/ +A114H/A122R/T126K/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/E184D/I205V/D206E/H210P/E214G/V223L/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/Y306F/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y/D428T/M437L 807/808R126C E16D/E26A/D29R/D33E/S49A/S58T/K91H/ + ++A114H/A122R/T126C/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/E184D/I205V/D206E/H210P/E214G/V223L/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/Y306F/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y/D428T/M437L 809/810R126T E16D/E26A/D29R/D33E/S49A/S58T/K91H/ ++ +++A114H/A122R/T126T/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/E184D/I205V/D206E/H210P/E214G/V223L/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/Y306F/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y/D428T/M437L 811/812R126S E16D/E26A/D29R/D33E/S49A/S58T/K91H/ +A114H/A122R/T126S/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/E184D/I205V/D206E/H210P/E214G/V223L/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/Y306F/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y/D428T/M437L 813/814R126W E16D/E26A/D29R/D33E/S49A/S58T/K91H/ ++ ++A114H/A122R/T126W/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/E184D/I205V/D206E/H210P/E214G/V223L/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/Y306F/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y/D428T/M437L 815/816R126M E16D/E26A/D29R/D33E/S49A/S58T/K91H/ + ++A114H/A122R/T126M/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/E184D/I205V/D206E/H210P/E214G/V223L/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/Y306F/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y/D428T/M437L 817/818R126E E16D/E26A/D29R/D33E/S49A/S58T/K91H/ ++A114H/A122R/T126E/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/E184D/I205V/D206E/H210P/E214G/V223L/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/Y306F/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y/D428T/M437L 819/820R126D E16D/E26A/D29R/D33E/S49A/S58T/K91H/ ++A114H/A122R/T126D/I128W/E130D/Y141F/A145G/F157Y/K158R/D180E/G183N/E184D/I205V/D206E/H210P/E214G/V223L/K246R/A256S/E270D/D272G/M286T/E298H/M302Q/Y306F/F322A/Y371F/G380R/Q383E/Q394G/ L412D/F413Y/D428T/M437L ¹Levels ofincreased activity were determined relative to the reference polypeptideof SEQ ID NO: 780, and defined as follows: “+” > 0.9; “++” > 1.1; and“+++” > 1.5.

Example 5: Comparison of Amylase Variant with and without His Tag

The C-terminus linker and His tag from SEQ ID NO: 810 was removed togenerate SEQ ID NO: 1758. Both these variants were grown in HTP and werescreened in duplicate for amylase activity at pH 6.8, activity post a 2hour gastric challenge (1.5 mg/mL pepsin, pH 2.3), a 2 hour intestinalchallenge, and a 2 hr protease challenge as described in Example 3.These two variants were found to have similar properties under thedifferent conditions screened, as shown by the absorbance value in Table5.

TABLE 5 Amylase activity comparisons between SEQ ID NO: 810 and SEQ IDNO: 1758¹ Activity Post Activity Post Intestinal Activity Post SEQ IDGastric Protease Protease NO: Activity Challenge Challenge Challenge(nt/aa) (A405) (A405) (A405) (A405)  809/810 4.1 ± 0.1 3.2 ± 0.2 3.3 ±0.1 3.6 ± 0.1 1757/1758 4.2 ± 0.1 3.4 ± 0.1 3.6 ± 0.2 3.6 ± 0.1 ¹Levelsof activity are expressed as absorbance units ± standard deviation at405 nm

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the invention.

What is claimed is:
 1. A recombinant amylase comprising a polypeptidesequence comprising at least 90% sequence identity to SEQ ID NO: 1728,wherein the polypeptide sequence comprises at least a substitution272G/K/P/R/S, 394E/G/N/R/S, or 91H, or any combinations thereof, whereinthe amino acid positions are relative to SEQ ID NO:
 2. 2. Therecombinant amylase of claim 1, wherein the polypeptide sequencecomprises SEQ ID NO: 156, 174, 276, 308, 422, 456, 546, 632, 664, 780,810, 1104, 1122, 1224, 1256, 1370, 1404, 1494, 1580, 1612, 1728, or1758.
 3. The recombinant amylase of claim 1, wherein said recombinantamylase exhibits at least one improved property selected from: i)enhanced catalytic activity; ii) increased tolerance to acidic pHconditions; iii) increased tolerance to at least one protease; iv)increased tolerance to at least one bile salt; v) increasedthermotolerance; or a combination of any of i), ii), iii), iv), and v),as compared to a reference amylase having the sequence of SEQ ID NO: 2or
 18. 4. A composition comprising at least one recombinant amylase ofclaim
 1. 5. A pharmaceutical composition comprising the recombinantamylase of claim 1, and a pharmaceutically acceptable carrier orexcipient.
 6. The recombinant amylase of claim 1, comprising apolypeptide sequence comprising at least 91% sequence identity to SEQ IDNO:
 1728. 7. The recombinant amylase of claim 1, comprising apolypeptide sequence comprising at least 95% sequence identity to SEQ IDNO:
 1728. 8. The recombinant amylase of claim 1, comprising apolypeptide sequence comprising at least 99% sequence identity to SEQ IDNO:
 1728. 9. The recombinant amylase of claim 1, wherein the polypeptidesequence comprises at least the substitution 272G, 272K, 272P, 272R, or272S.
 10. The recombinant amylase of claim 1, wherein the polypeptidesequence comprises at least the substitution 394E, 394G, 394N, 394R, or394S.
 11. The recombinant amylase of claim 1, wherein the polypeptidesequence comprises at least the substitution 91H.
 12. The recombinantamylase of claim 1, wherein the polypeptide sequence comprises at leastthe substitution 272G, 394G, or 91H, or any combinations thereof. 13.The recombinant amylase of claim 1, comprising a polypeptide sequencecomprising at least 92% sequence identity to SEQ ID NO:
 1728. 14. Therecombinant amylase of claim 1, comprising a polypeptide sequencecomprising at least 93% sequence identity to SEQ ID NO:
 1728. 15. Therecombinant amylase of claim 1, comprising a polypeptide sequencecomprising at least 94% sequence identity to SEQ ID NO:
 1728. 16. Therecombinant amylase of claim 1, comprising a polypeptide sequencecomprising at least 96% sequence identity to SEQ ID NO:
 1728. 17. Therecombinant amylase of claim 1, comprising a polypeptide sequencecomprising at least 97% sequence identity to SEQ ID NO:
 1728. 18. Therecombinant amylase of claim 1, comprising a polypeptide sequencecomprising at least 98% sequence identity to SEQ ID NO: 1728.